Advances in Genetics, volume 56

Volume 56 Advances in Genetics Advances in Genetics, Volume 56 Serial Editors Jeffery C. Hall Waltham, Massachuset...

Author: Jeffrey C. Hall | Jay C. Dunlap | Theodore Friedmann | Veronica van Heyningen

234 downloads 1259 Views 3MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

Volume 56

Advances in Genetics

Advances in Genetics, Volume 56

Serial Editors

Jeffery C. Hall Waltham, Massachusetts

Jay C. Dunlap Hanover, New Hampshire

Theodore Friedmann La Jolla, California

Veronica van Heyningen Edinburgh, United Kingdom

Volume 56

Advances in Genetics Edited by

Jeffrey C. Hall Department of Biology Brandeis University Waltham, Massachusetts

Jay C. Dunlap Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire

Theodore Friedmann Center for Molecular Genetics University of California at San Diego School of Medicine La Jolla, San Diego, California

Veronica van Heyningen MRC Human Genetics Unit Western General Hospital Edinburgh, United Kingdom

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Contents Contributors

vii

1 Biological Activity and Biotechnological Aspects of 1 Peptide Nucleic Acid Karin E. Lundin, Liam Good, Roger Stro¨mberg, Astrid Gra¨slund, and C. I. Edvard Smith I. II. III. IV. V.

Introduction 2 PNA: Chemistry and Structure 4 Biological Activity 13 Biotechnological Aspects of PNA 29 Concluding Remarks 38 References 38

2 Changing Images of the Gene

53

George P. Re´dei, Csaba Koncz, and Jane D. Phillips

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV.

Introduction 54 The Hypothetical Gene 55 Physical Concepts of the Gene 57 Chemical Nature of the Gene 59 The Genetic Code 63 Structure of the Gene 64 Molecular Units of the Gene 68 Higher Orders of Genes 70 Gene Number and Gene Size 73 Genes in the Cytoplasm 75 Epigenetics 78 Prions 83 Conclusions and Outlook 86 Epilog 87 References 88

v

Contents

vi

3 Historical and Modern Genetics of Plant 101 Graft Hybridization Yongsheng Liu

I. II. III. IV. V. VI. VII. VIII.

Introduction 102 Historical Background 103 The Existence of Graft Hybrids 107 Methods of Graft Hybridization 114 Characteristics of Graft Hybridization 115 Mechanisms Underlying Graft Hybridization Significance of Graft Hybridization 120 Conclusions 124 References 125

4 Step into the Groove: Engineered Transcription Factors as Modulators of Gene Expression

116

131

Astrid E. Visser, Pernette J. Verschure, Willemijn M. Gommans, Hidde J. Haisma, and Marianne G. Rots I. Introduction 132 II. Transcriptional Therapy 134 III. Engineered Zinc-Finger–Based Transcription Factors (ZF-TFs) and the Influence of Nucleosomes IV. Concluding Remarks and Future Perspectives 155 References 156

148

5 Step out of the Groove: Epigenetic Gene Control Systems 163 and Engineered Transcription Factors Pernette J. Verschure, Astrid E. Visser, and Marianne G. Rots I. Influence of Epigenetic Mechanisms on Gene Expression 164 II. How Do ZF-TFs Modulate Epigenetic Gene Regulation? 182 III. Epigenetic Aspects to Consider for ZF-TF Approaches 191 References 196

Index

205

vi

Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Willemijn M. Gommans (131) Therapeutic Gene Modulation, Groningen University Institute for Drug Exploration, University of Groningen, The Netherlands Liam Good (1) Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius vag 35, 171 77 Stockholm, Sweden Astrid Gra¨slund (1) Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden Hidde J. Haisma (131) Therapeutic Gene Modulation, Groningen University Institute for Drug Exploration, University of Groningen, The Netherlands Csaba Koncz (53) Max-Planck-Institut, D-59829 Ko¨ln, Germany Yongsheng Liu (101) Department of Horticulture, Henan Institute of Science and Technology, Xinxiang 453003, China Karin E. Lundin (1) Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden Jane D. Phillips (53) Life Sciences Center, University of Missouri, Columbia, Missouri 65211 George P. Re´dei (53) University of Missouri, Columbia, Missouri 65203 Marianne G. Rots (131, 163) Therapeutic Gene Modulation, Groningen University Institute for Drug Exploration, University of Groningen, The Netherlands C. I. Edvard Smith (1) Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital, Huddinge, 141 86 Stockholm, Sweden Roger Stro¨mberg (1) Department of Biosciences, Karolinska Institutet, Novum, 141 57 Huddinge, Sweden Pernette J. Verschure (131, 163) Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, 1098SM Amsterdam, The Netherlands Astrid E. Visser (131, 163) Department of Molecular Genetics, Leiden Institute of Chemistry, University of Leiden, 2300 RA Leiden, The Netherlands; Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, 1098SM Amsterdam, The Netherlands vii

This page intentionally left blank

1

Biological Activity and Biotechnological Aspects of Peptide Nucleic Acid Karin E. Lundin,* Liam Good,† Roger Stro¨mberg,‡ Astrid Gra¨slund,§ and C. I. Edvard Smith* *Department of Laboratory Medicine, Clinical Research Center Karolinska Institutet, Karolinska University Hospital, Huddinge 141 86 Stockholm, Sweden † Center for Genomics and Bioinformatics, Karolinska Institutet Berzelius vag 35, 171 77 Stockholm, Sweden ‡ Department of Biosciences, Karolinska Institutet, Novum 141 57 Huddinge, Sweden § Department of Biochemistry and Biophysics, Arrhenius Laboratory Stockholm University, 106 91 Stockholm, Sweden

I. Introduction II. PNA: Chemistry and Structure A. Synthesis of PNA B. Solution properties of PNA C. PNA–nucleic acid complexes: Formation and structures D. Variations on a theme: PNA analogs and conjugates III. Biological Activity A. Cellular uptake B. PNA effects on RNA C. PNA in gene regulation D. Future prospects for PNAs as antisense and antigene drugs IV. Biotechnological Aspects of PNA A. PNA-anchors acting as genetic “glue” B. PNA beacons and other fluorescent PNA-probes C. Other biotechnological applications V. Concluding Remarks Acknowledgments References Advances in Genetics, Vol. 56 Copyright 2006, Elsevier Inc. All rights reserved.

0065-2660/06 $35.00 DOI: 10.1016/S0065-2660(06)56001-8

2

Lundin et al.

ABSTRACT During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptidelike backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at lowion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter. ß 2006, Elsevier Inc.

I. INTRODUCTION Biochemists have long been searching for new synthetic compounds with the potential to interact with nucleic acids in a stable and sequence-specific manner. The desired compounds should possess a capacity to hybridize to DNA and RNA with high affinity while still displaying sufficient specificity to distinguish singlebase mismatches. During the past decade several hundred different analogs, containing natural nucleobases on a modified backbone, have been synthesized. A pioneering example of this is peptide nucleic acid (PNA), an analog with a noncyclic peptide-like backbone. The original aminoethylglycine-based PNA (Fig. 1.1) was first described by Peter Nielsen, Michael Egholm, Rolf Berg and Ole Buchardt in 1991 (Buchardt et al., 1992; Nielsen et al., 1991). Using molecular modeling the Danish group removed the sugar phosphate backbone and replaced it with a polyamide of N-(2-aminoethyl)glycine. This resulted in an oligonucleotide analog with an uncharged backbone that is achiral (i.e., does not have stereoisomers). Owing to its flexible and neutral backbone PNA displays very good hybridization properties at “low” as well as “high” ionic strength, and has subsequently attracted large interest both in biotechnology and biomedicine. The unnatural backbone also provides a high biostability (Demidov et al., 1994), further increasing the possibility to use PNA for in vivo applications. Peptide nucleic acid has the capacity to act both as antisense and antigene

1. Biological and Biotechnological Aspects of PNA

3

Figure 1.1. The basic structural difference and similarity between DNA and PNA.

agents, as well as to anchor biological active molecules to DNA in a sequencespecific manner. However, presumably due to the lack of charges in the backbone, PNA does not diffuse into intact cells, and a number of different modifications have consequently been made in order to increase cellular uptake, that is, chemical modifications in the PNA itself or conjugation with positively charged peptides and dyes. In this regard, the possibility to induce cell-specific uptake is another important issue that has been addressed in order to increase the potential for in vivo applications. Peptide nucleic acid has been used in a number of diagnostic applications, which often depend on its capacity to hybridize to natural nucleic acid oligomers independent of salt concentration. Furthermore, several diagnostic applications exploit the efficient discrimination of single-base mismatches. Examples of this are the identification of single nucleotide polymorphisms with blocking of PCR amplification of the background gene using a PCR clamp (Ørum, 2004), or by real time PCR using a “light-up” PNA-probe (Kuhn et al., 2002, 2001; Seitz, 2000). Similarly, rapid detection of the chromosomal localization of specific genes by fluorescence in situ hybridization (FISH) using fluorophore labeled PNAs are now possible (Nielsen et al., 2004; Pellestor et al., 2005). Under certain conditions PNA also is able to strand-invade into doublestranded DNA, a capacity that is utilized for sequence-specific labeling and binding of functional molecules to both genomic and plasmid DNA, areas that are also included in this chapter.

4

Lundin et al.

II. PNA: CHEMISTRY AND STRUCTURE Peptide nucleic acids can be described as unnatural polypeptides with side chains containing the heterocyclic bases found in nucleic acids. The polyamide backbone imposes distances between the nucleobases that are similar to those found in natural DNA and RNA (Fig. 1.1). This enables interactions with nucleic acids, such as Watson–Crick base pairing, while the uncharged backbone provides several advantageous properties (see in a later section).

A. Synthesis of PNA The assembly process of PNA is virtually identical to peptide synthesis. This is also one of the strengths of PNA technology, that is, that PNA can be readily made by conventional automated solid phase synthesis using commercial peptide synthesizers. The elongation of the PNA oligomer takes place through condensation of the carboxy function of the building block with the deprotected amino function of the growing chain, aided by a condensing agent (Scheme 1.1).

Scheme 1.1. Schematic presentation of solid phase synthesis of PNA. (For Prot1 and Prot2 see Fig. 1.2, HBTU ¼ O-(benzotriazol-1-yl)-N, N, N0 , N0 -tetramethyluronium hexafluorophosphate, HATU ¼ O-(7-azabenzotriazol-1-yl)-N, N, N0 , N0 -tetramethyluronium hexafluorophosphate).

1. Biological and Biotechnological Aspects of PNA

5

The same condensing agent and a cycle with only minor alterations from standard peptide synthesis can be used (Beck, 2002; Coull et al., 1996; Dueholm et al., 1994; Thomson et al., 1995). Synthesis of oligomers with all four natural bases was initially carried out using tert-butoxyarbonyl (Boc) protection on the aminoterminal, and benzyloxycarbonyl (Cbz) protection (1a in Fig. 1.2) on the exocyclic amino functions of the nucleobases, in a procedure very similar to peptide synthesis with Boc-protected amino acids (Dueholm et al., 1994). Later on, the fluorenylmethoxycarbonyl (Fmoc) strategy, so commonly used in peptide synthesis, has been introduced both with Cbz (Fig. 1.2, 1b) (Thomson et al., 1995) and more labile benzhydryloxycarbonyl (Bhoc, Fig. 1.2, 1d) (Coull et al., 1996) and monomethoxytrityl (MMT, Fig. 1.2, 1e) (Breipohl et al., 1996) base protection. In addition, acid labile MMT protection for the amino function has been used in combination with N-acyl protection on nucleobases and is particularly suited to synthesis of PNA–DNA chimera (Finn et al., 1996; Uhlmann et al., 1996; van der Laan et al., 1995). Custom made PNA with the four common bases is commercially available, but at an expense that cannot be neglected, especially for more extensive studies. This probably limits the spread of PNA technology somewhat. An alternative is to make PNA from commercially available or, for extensive use, self-made building blocks. The building blocks are typically synthesized via one of two generally used routes (Scheme 1.2A). The aminoethylglycine backbone is first assembled either through alkylation of a protected ethylenediamine with an alkyl bromoacetate (Clivio et al., 1998; van der Laan et al., 1996) or through reductive amination of N-protected aminoacetaldehyde with a glycine ester (Farese et al., 1996; Salvi et al., 1994). The protected nucleobases are then

Figure 1.2. Common building blocks for PNA synthesis with indicated protection of aminoterminal and exocylic aminofunction of the bases. Base ¼ T, C, A, and G; Boc ¼ tert-butoxycarbonyl; Cbz ¼ benzyloxycarbonyl; Fmoc ¼ 9-fluorenylmethoxycarbonyl; Bhoc ¼ diphenylmethoxycarbonyl; MMT ¼ (4-methoxyphenyl)diphenylmethyl; Acyl ¼ benzoylderivatives for A and C, and isobutyryl for G.

6

Lundin et al.

Scheme 1.2. Synthesis of PNA building blocks. For Prot1 and Prot2 see Fig. 1.2, R ¼ H, methyl, ethyl, or t-butyl; TFA ¼ trifluoroacetic acid; DCC ¼ dicyclohexylcarbodiimid; HBTU ¼ O-(benzotriazol-1-yl)-N, N, N0 , N0 -tetramethyluronium hexafluorophosphate.

converted to N-alkyl acetic acid derivatives by reaction with bromoacetic acid or bromoacetic acid esters followed by hydrolysis (Scheme 1.2B). The backbone and base components are then linked by formation of an amide (Scheme 1.2C).

B. Solution properties of PNA Peptide nucleic acid is a neutral and relatively hydrophobic molecule, and consequently its behavior in aqueous solutions may cause some experimental problems. Peptide nucleic acid solubility in HEPES buffer at pH 7.3, 37 C,

1. Biological and Biotechnological Aspects of PNA

7

has been reported as 0.1–0.5 mM (Noble et al., 1995). Although this suggests fairly good solubility, single-stranded PNA tends to aggregate in aqueous solution, forming discrete structural species that can be observed by, for example, 1 H NMR (Leijon et al., 1994). One can even observe “melting” processes of single-stranded PNA in aqueous solution (Tomac et al., 1996). Adding complex-forming DNA or RNA strands to the single-stranded PNA dissolves the PNA aggregates and leads to well-defined complex structures, which will be discussed in a later section. In the more recent applications, charged amino acid residues, such as lysines, are usually added to the PNA sequences, and this greatly decreases experimental problems regarding solubility and aggregation. For PNA to be used in antisense or antigene applications, the cellular delivery problem must be addressed. This is a more serious problem than solubility and has prompted a variety of strategies to improve uptake, as summarized in the review by Nielsen (2003). The different methods applied include direct delivery of unmodified PNA to the cell-culture medium, physical disruption of cell membranes by electroporation, delivery via cationic liposomes, and conjugation with small cationic peptides (so called cell-penetrating peptides, CPPs) that mediate translocation across cell membranes. The CPP-based approaches are particularly interesting, and CPPs can be attached to the PNA sequence either by conjugation or continuous synthesis.

C. PNA–nucleic acid complexes: Formation and structures

1. Three-dimensional structures Peptide nucleic acid was originally designed to bind as a third strand to a DNA double helix and thereby to modify its properties in transcription or translation. Experimental evidence soon showed that the structural versatility of PNA– nucleic acid complexes was much more varied. Depending on sequence and experimental conditions, PNA–nucleic acid duplexes or (PNA)2–nucleic acid triplexes can be formed. The nucleic acid can be either DNA or RNA. Due to the charge neutrality of PNA, the PNA–nucleic acid complexes are generally more temperature stable than the corresponding pure nucleic acid complexes, particularly at moderate ionic strengths (Dueholm et al., 1994). A PNA–PNA complex is the most stable among the complex variants. A general observation regarding heteroduplexes involving DNA or RNA is that the nucleic acid strand guides PNA to adopt a structure that allows the DNA or RNA to remain in a B-like and A-like conformation, respectively, as shown by early NMR structure studies in solution (Beck, 2002; Brown et al., 1994; Eriksson and Nielsen, 1996). PNA–RNA duplexes are of principal interest because of potential use of PNA in antisense strategy. However, PNA binding to a complementary sequence in mRNA is not entirely predictable,

Lundin et al.

8

Table 1.1. Helical Parameters for Typical B-DNA, A-RNA and a PNA–PNA Double Helix Helix

˚) X-displacement (A

Inclin. ( )d

Twist ( )

˚) Rise (A

Base tilt ( )

Bases/turn

B-DNAa A-RNAb P-PNAc

0.0 –5.3 –8.3

1.5 15.8 0.3

36 32.7 19.8

3.4 2.8 3.2

0.0 0.0 1.0

10 11 18

a Neidle, S., ed. (1999). “Oxford Handbook of Nucleic Acid Structures.” Oxford University Press, New York. b Pooga et al. (1998). c From a selfcomplementary tetramer region of four basepairs in the middle of a decamer duplex, Leijon et al. (1994). d ( ) ¼ degrees.

and the PNA–RNA duplex is not a substrate for RNaseH (Knudsen and Nielsen, 1996). Crystal structures of PNA homoduplexes, heteroduplexes, and heterotriplexes show that PNA prefers a P-form helix, different from both A- and B-forms of RNA and DNA. Table 1.1 summarizes some of the structural features of ideal B- and A-form helices together with the P-form observed in a PNA–PNA hexamer (Rasmussen et al., 1997). A crystal structure of a partly self-complementary PNA oligomer shows a complex duplex–triplex network (Petersson et al., 2005), illustrating a very high adaptability of the PNA backbone in response to nucleobase stacking and hydrogen-bonding possibilities.

2. Processes leading to PNA complex formation with duplex DNA: Strand invasion Interaction of single-stranded PNA with double-stranded DNA is of interest not only because of potential applications in antigene strategy but also for the possible use of PNA in different biotechnological applications (as discussed in a later section). Various modes of interactions are possible, depending on sequences and conditions. Figure 1.3 illustrates the triplex and duplex invasion modes, in which the targeting PNA breaks up the double helix and complexes with the complementary DNA strand. Duplex invasion can result if the target DNA is a homopyrimidine and the PNA has a homopurine sequence. If instead the DNA target is a homopurine sequence, a very stable triplex invasion complex can form. Synthesizing pyrimidine-PNAs as a palindrome sequence with a flexible linker in the middle creates a so called bisPNA or “PNA clamp,” allowing triplex formation in optimal directions with the Watson–Crick basepairing in an antiparallel and Hoogsten bonding in a parallel orientation (Fig. 1.3) (Egholm et al., 1995; Kuhn et al., 1999). The triplex invasion complex

1. Biological and Biotechnological Aspects of PNA

9

Figure 1.3. Models of some PNA invasion processes into double-stranded DNA. The PNA strand is shown with a black backbone. (A) PNA–DNA2 triplex, (B) duplex invasion with homopyrimidine target, (C) triplex invasion with homopurine target, (D) triplex invasion by bisPNA clamp, and (E) dubble duplex invasion to mixed-base targets by pseudocomplementary PNAs.

is, however, very slow to form at neutral pH, unless the cytosines in the “Hoogsteen-pairing” strand are exchanged to pseudo-isocytosines, also called J bases (Egholm et al., 1995). For practical applications, the rate of formation can be increased by using cationic PNAs (e.g., with extra lysines linked) or by linking the PNA to an intercalating molecule such as 9-aminoacridine (Kuhn et al., 1998).

D. Variations on a theme: PNA analogs and conjugates The interesting properties and many applications of the original aminoethylglycine PNA (aeg-PNA) have stimulated development of a large number of PNA modifications, including replacing glycine with a chiral amino acid

10

Lundin et al.

(Ganesh and Nielsen, 2000). However, the majority of modified PNAs involve additional synthesis steps and typically lower affinities relative to the original aeg-PNA, especially for the early attempts on modifications. It seems that by the first design a good balance between flexibility and rigidity was found, so that PNA can adopt to both DNA and RNA and without too high-entropy cost. There are, however, PNA-variants that do stand out and induce properties that give interesting interactions with, or improved affinity for, nucleic acids. Particularly interesting are designs that introduce further conformational restriction to PNA. One example is the introduction of 4-aminoproline-based residues (4 in Fig. 1.4), the chirality of which strongly influences PNA properties (Gangamani et al., 1999a,b, 1996; Jordan et al., 1997a,b). Perhaps most interesting is that introduction of a single 4-amino proline residue into aeg-PNA led to stabilization of PNA–DNA hybrids and also to directionality in binding orientation. However, homo-oligomers with only 4-aminoproline residues did not form stable duplexes (Gangamani et al., 1996; 1999a,b). An example of another conformationally restricted modification, that when fully introduced failed to form stable duplexes, is pyrrolidine-methyl-thymine-1-acetyl-glycinePNA (pmg-PNA, 5 in Fig. 1.4) (Slaitas and Yeheskiely, 2001, 2002). Upon introduction of one and two residues containing this modification, a high preference for RNA binding over DNA binding was found, especially when introducing two neighboring R-isomers. This was, however, at the cost of overall stability of the duplex. Another example of a PNA-modification that has a high preference for RNA but forms highly stable hybrids is the cyclohexyl-PNA (ch-PNA, 6 in Fig. 1.4) (Govindaraju et al., 2003, 2004c). For DNA target recognition, the aminoethylpropyl-PNA (aep-PNA, 7, Fig. 1.4) appears promising (D’Costa et al., 1999). This modification, which also introduces positive charges in the backbone, gives more stable duplexes with DNA without loss of selectivity and is, as a bonus, also more soluble in aqueous media. An example of a modification containing two backbone ring structures per base residue is the proline-based modification (8; Fig. 1.4), which displayed high-DNA affinity and mismatch discrimination (Vilaivan and Lowe, 2002). Another highly promising design is the introduction of a cyclopentyl ring in the PNA backbone (cp-PNA, 9 in Fig. 1.4). The cis-isomers gives stabilization of triplexes (Govindaraju et al., 2004a,b), and the (S,S,)-trans-isomer gives highly stabilized duplexes (Myers et al., 2003; Pokorski et al., 2004). The (S,S,)-transisomer seems most promising for DNA recognition, as substitution of a few standard aeg-PNA units with this modification in a mixed sequence gives not only five to eight degrees increase in affinity per inserted cp-PNA unit but also a greatly increased mismatch discrimination (Myers et al., 2003; Pokorski et al., 2004). It will be interesting to see how this modification performs when used in

1. Biological and Biotechnological Aspects of PNA

Figure 1.4. Schematic representation of different PNA analogs.

11

12

Lundin et al.

diagnostic applications. Additional conformation-restricted PNA analogs with interesting properties have been developed, and their affinity and selectivity generally display a complex dependence on structural differences, chirality, and sequence (Govindaraju and Kumar, 2005; Govindaraju et al., 2005; Kitamatsu et al., 2004; Kumar and Ganesh, 2005; Pokorski et al., 2005). Examples of other modifications are the fluorinated olefin PNA (F-OPA; Hollenstein and Leumann, 2003, 2005), various metal binding PNAs (Mokhir et al., 2003a, 2004a,b; Popescu et al., 2003), aromatic backbones (Fader and Tsantrizos, 2002; Fader et al., 2001, 2004; Tsantrizos et al. 1997), alphahelical peptide nucleic acids (Garner et al., 1999, 2000, 2001; Huang et al., 2004), and cationic PNAs. The latter includes incorporation of guanidine functionalities in the backbone (Barawkar and Bruice, 1999; Barawkar et al., 2000), as well as partial replacement of glycine within aeg-PNA with positively charged amino acids such as arginine (Zhou et al., 2003) and lysine (Menchise et al., 2003; Sforza et al., 1999, 2000). Several of these recently developed modifications display interesting properties, but affinity and selectivity, although good in several cases, is in general less spectacular than for some of the conformationally restricted analogs. Among the various attempts to develop more functional PNAs through conjugation, peptide attachments appear to be particularly interesting. Peptide nucleic acid-peptide conjugates can either be assembled by on-line solid phase synthesis or by fragment ligation. This has been reviewed by de Koning et al. (2003). Other conjugates include linking to sugars (Hamzavi et al., 2003a) or aminoglycosides (Riguet et al., 2004), fatty acids (Vernille et al., 2004) or steroids (Rebuffat et al., 2002), polyamines (Gangamani et al., 1997; Petersen et al., 2004b; Verheijen et al., 2000), and different aromatics (Ikeda et al., 2001, 2002; Mokhir et al., 2003b; Ross et al., 2003), most of which increase the binding affinity to DNA. In particular, conjugate with an acridine derivative more readily undergoes strand invasion (Bentin and Nielsen, 2003), while the Hoechst minor groove binder (Nielsen et al., 2005) provides an additional handle by which selectivity can be modified. There are also interesting modifications that can be considered at the borderline of calling them PNA-modifications, such as replacement of the amide linkage by a phosphonate linkage (Peyman et al., 1996; van der Laan et al., 1996), which is really not a peptide or an amide. Amide linked oligonucleotides can, however, be called polyamide nucleic acids, as peptide nucleic acids were first called, but not peptidic. These molecules are interesting because even as partially modified oligonucleotides they provide substantial duplex stabilization (De Mesmaeker et al., 1994; Nina et al., 2005; Rozners et al., 2003), but there is still some synthetic work left to be done before efficient synthesis of fully modified oligonucleotides with all bases can be made.

1. Biological and Biotechnological Aspects of PNA

13

III. BIOLOGICAL ACTIVITY Peptide nucleic acid is very resistant to nuclease degradation (Demidov et al., 1994) and exhibits little or no binding to serum proteins. Therefore, PNA overcomes two of the major complications for the use of traditional oligonucleotides in biological applications. In addition, PNA display a high stability toward proteases (Demidov et al., 1994), further increasing its potential within the biomedical and biotechnological field.

A. Cellular uptake Passive diffusion of PNA over lipid membranes is very slow and has been attributed to the neutral charge (Wittung et al., 1995). Synthesizing PNA oligomers as peptide chimeras, or conjugating them to charged or lipophilic molecules, are two approaches used to increase uptake into both eukaryotic and prokaryotic cells, see Table 1.2.

1. Uptake of PNA in eukaryotic cells a. Cellular uptake of unmodified PNA in vitro Due to the very slow diffusion rate over lipid membranes, nonreceptor mediated uptake of unmodified PNA into eukaryotic cells is generally not observed, and there are only a few reports of biological effects following free delivery in cell culture. Sei et al. (2000) reported effects on viral production after simply adding PNAs directly to the cell-culture medium of an HIV-1 infected lymphoma cell line. However, the concentrations of PNA used by these investigators were very high, 20 mM. In addition, a significant antisense effect has been reported in primary neurons and astrocytes cultured in presence of 5–10 mM biotinylated, but otherwise unmodified, antisense PNA (Adlerz et al., 2003).

b. Cellular uptake of unmodified PNA in vivo Several groups have also reported biological effects of PNA administration in vivo, after direct intracerebral (Tyler et al., 1998), intrathecal (Rezaei et al., 2001), or intraventricular (Fraser et al., 2000; McMahon et al., 2003) injections, as well as after systemic or intraperitoneal administration (McMahon et al., 2002b; Tyler et al., 1999) in rats. The reported capacity of unmodified PNAs to cross the blood–brain barrier is potentially very interesting, but whether this is a common feature of unmodified PNAs remains to be confirmed. Pardridge et al. (1995) reported the uptake of biotinylated PNA in the brain to be negligible after intravenous administration, unless conjugated to a transferrin receptorbinding mouse monoclonal antibody. Notably, most in vivo reports on direct

Table 1.2. Cellular Delivery of PNA to Eukaryotic Cells Ex Vivo

PNA modification and labeling

Delivery method

PNA concentration (mM)

Cell type

Detection system

References

Mba-15-mer unmodified or fluorescein labeled

Direct uptake

20 30

HIV-infected H9 cells

Antisense (viral antigen)/ IF and FACS

(Sei et al., 2000)

Mb-21-mer-transportan or pAnt,þbiotin

CPPb-mediated

1

Bowes melanoma

(Pooga et al., 1998).

Mb-17-mer-pkkkrkvc þ/–TAMRAd

CPP-mediated

10

Burkitts lymphoma

Mb-18-mer-kkkk

CPP-mediated

1–10

Mb-13-mer-g4cskcc radioactivity or fluorescein Mb-13-mer-lactose8, þ/ rhodamine

IGF1e-receptor mediated

1

HeLa-S3-EGFP with aberrant splicing P6 (human IGF1-R expressing 3T3 cells)

IF (avidin conj) and antisense (protein expression) IF and antigene activity (myc-mRNA/protein; cell viability) Splice correction (FACS and RT-PCR) 14 C uptake and IF

ASGPf receptor mediated

1 6–20

HepG2 (hepatoblastoma cell line)

IF, antisense (telomerase inhibition)

(Cutrona et al., 2000)

(Sazani et al., 2001) (Basu and Wickstrom, 1997) (Zhang et al., 2001)

MB-13-mer, þ/ rhodamine

DNA and cationic liposomes

1

DU145 (prostate-tumor) and 293 cells

Mb-18-mer Acrgþ/ fluorescein

Cationic liposomes (þ/ DNA)

1–2

Mb-11-mer TPPhconjugate with biotin bisPNA with lysines and fluorescein

Lipophilic ion mediated uptake Transient membrane permeabilization using SLOi Electroporation

1

JAR, HeLa-pLuc705 with aberrant splicing 143B (osteosarcoma), human fibroblasts 3340 cells (mouse fibroblast)

bisPNA a

1

2–5

Mixed-base PNA. Cell-penetrating peptide. c Amino acids in peptides indicated in lower case single letter code. d N, N, N0 , N0 -tetramethyl-6-carboxy-rhodamine ester. e Insuline like growth factor 1. f Asialoglycoprotein receptor. g 9-Aminoacridine-conjugate. h Triphenylphosphoium cation. i Streptolysin O. b

K562

IF and FACS and antisense (telomerase inhibition) IF/splice correction (enzyme activity and RT-PCR) Immunoblotting and IF

(Hamilton et al., 1999)

(Shiraishi and Nielsen, 2004)

IF and FACS

(Muratovska et al., 2001) (Faruqi et al., 1998)

Increased gene expression

(Wang et al., 1999)

16

Lundin et al.

cellular uptake of unmodified PNAs are related to antisense or antigene functions for proteins expressed in the central nervous system. This might indicate a special uptake mechanism in these cells. The tissue distribution and pharmacokinetic parameters for an unmodified PNA, after intravenous injections in rat, has been reported (McMahon et al., 2002a). Two hours after injection the organ distribution was investigated using a gel shift assay. Peptide nucleic acid was found at low levels in all organs examined—kidney, liver, heart, brain, and spleen, with the highest concentration found in kidneys. The distribution half-life was found to be 3 (þ/–3) min and the elimination half-life 17 (þ/–3) min. The total plasma clearance was 3.4 (þ/–0.9) ml/min, and approximately 90% of the PNA was recovered as intact molecules in the urine within 24 h after administration. This confirms the high biostability reported for PNA after incubation in serum and cell-lysates in vitro (Demidov et al., 1994).

c. Uptake of PNA conjugated to cell-penetrating peptides In many studies, PNA has been conjugated to peptides with known cellpenetrating activity (CPPs), such as pTat from HIV (Vives et al., 1997), penetratin from the Drosophila protein Antennapedia (Derossi et al., 1994, 1998), and transportan (Pooga et al., 1998). By either the continuous synthesis of PNAs as peptide chimeras or by chemically linking the PNA to already synthesized peptides, increased cellular uptake could be detected (Kaushik et al., 2002; Koppelhus et al., 2002; Pooga et al., 1998; Thierry et al., 2003). In some studies, cellular uptake was detected at culture concentrations as low as 500 nM. Cell-penetrating activities have been reported to display capacity to transport a cargo over the cell membrane in an energy and receptor independent manner. The mechanisms of translocation are not completely understood, despite intense studies; endocytotic mechanisms as well as more direct membrane interaction mechanisms may contribute (Belting et al., 2005; Joliot and Prochiantz, 2004; Kaplan et al., 2005). Especially studies prior to 2003 must be carefully interpreted, as the fixation methods used can introduce microscopy artifacts (Lundberg and Johansson, 2001; Richard et al., 2003). Due to these findings, microscope evaluations of cell uptake are now generally performed on live cells. Chaubey et al. (2005) described a concentration dependent, rapid, and energy independent uptake of a TAMRA labeled PNA-transportan conjugates into several different cell lines. Also, uptake was demonstrated after pretreatment of cells with phenylarsine oxide, which blocks both endocytosis and receptor functions. This study reported, furthermore, inactivation of a pseudotyped HIV-1 virus after preincubation of viral particles with the anti-TAR PNA-transportan conjugate at a concentration as low as 100 nM (Chaubey et al., 2005). Whether this effect was mediated by virus-membrane penetrating PNA or by PNA attached on the viral surface, gaining access to viral RNA only

1. Biological and Biotechnological Aspects of PNA

17

after cellular infection, was not clearly elucidated, however. Also, simply synthesizing the PNA as a chimera with a highly basic peptide, like the classic SV40 large T antigen nuclear localization signal (NLS) (Cutrona et al., 2000) or multiple lysine residues (Sazani et al., 2001), seems to be enough to promote increased cellular uptake in vitro, at least at concentrations above 3–5 mM. The efficiency of the CPP strategy seems to depend on the target cell line as well (Koppelhus et al., 2002). In order to verify intracellular uptake several studies have used biological assays, detecting changes in cell growth as the readout, while other have used directly fluorescence-labeled PNAs to measure uptake. Using labeled PNAs, Koppelhus et al. (2002) reported the occurrence of unspecific toxic reactions at PNA concentrations above 5–10 mM. Kaihatsu et al. (2004) demonstrated cellular uptake of rhodamine labeled antisense PNA-cationic peptide conjugates at a concentration as low as 200 nM but had to use 50-fold higher concentrations to downregulate protein expression.

d. Uptake of PNA targeted to specific cellular receptors Other groups have chosen the strategy of coupling PNA to antibodies or ligands, binding to specific cellular receptors. Examples of targeted receptors are the transferrin receptor (Pardridge et al., 1995), the insulin-like growth factor 1 receptor (IGF1-R) (Basu and Wickstrom, 1997), and the liver-specific ascialo glycoprotein receptor (ASGP-R) (Zhang et al., 2001). The targeting approach has the advantage of directing PNA delivery to specific cell types. Unfortunately, PNA by itself does not provide mechanisms mediating endosomal release following receptor-mediated uptake, and subsequently the PNA is almost exclusively localized in cytoplasmic vesicles, resulting in very weak biological effects. One way to possibly solve this problem is to attach endosome-breaking peptides via disulfide bridges that will be reduced once inside the cell, or via peptide linkers containing specific amino-acid sequences that will be cleaved by cellular proteases in the endosome (Svahn et al., 2004b). When released from the PNA, these peptides will induce endosomal rupture and release PNA into the cytoplasm.

e. Uptake via liposomes and lipophilic conjugates The uncharged nature of the PNA-molecule has made it difficult to create the stable complexes needed to transport PNA into cells using traditional transfection protocols involving cationic liposomes or polymers. By hybridizing PNA to a partly complementary “carrier” DNA, the use of liposomes has, however, been successful (Hamilton et al., 1999). Kaihatsu et al. (2004) compared the direct addition of PNA-cationic peptides to cell cultures with DNA–lipid mediated uptake. Lipidmediated delivery required 40–50 times lower PNA concentrations to achieve a similar biological effect in immortal cell lines. When added to primary cells, the lipid formulation, however, frequently induced extensive cell death. This was not seen when adding PNA-peptides alone (Kaihatsu et al., 2004).

18

Lundin et al.

Figure 1.5. Peptide nucleic acid conjugated to lipophilic cations are readily transported over the cell membrane and due to the high membrane potential it assembles in mitochondria. When conjugation is performed via a disulphide bridge, enzymes in the cytoplasm will reduce the s–s bond and free PNA will remain in the cytosol.

Peptide nucleic acid conjugated to the DNA-intercalating dye 9-aminoacridine (Acr-PNA) has been successfully transfected into cells with use of lipofectamine, also without the addition of carrier DNA (Shiraishi and Nielsen, 2004). Coupling PNA to the lipophilic triphenylphosphonium cation (TPP) has also been reported. Owing to the negative membrane potentials TPPPNA was shown to accumulate in mitochondria (Muratovska et al., 2001). By conjugating thiol-TPP to PNA via a reducible disulfide bond formation (Filipovska et al., 2004), PNA was able to accumulate in the cytosol, as illustrated in Fig. 1.5.

2. Cellular delivery into bacteria Bacteria are well protected against foreign chemicals by thick cell barriers that are composed of a low-permeability cell wall. The composition and structure of bacterial cell walls differ between classes of microbes but are similar in providing stringent protection, with a molecular weight cut off of about 1000 g/mol (Nikaido, 2003). Furthermore, pathogenic bacteria often harbor in host cells, providing added protection. For example, many pathogens occupy host vesicles or form biofilms that provide added protection. Therefore, bacteria pose challenging targets for antisense PNAs, which are much larger than conventional antibiotics. Fortunately, attaching PNAs to carrier peptides can help solve the cellular delivery problem. Bacterial surfaces typically contain negatively charged

1. Biological and Biotechnological Aspects of PNA

19

lipids, and certain cationic peptides are electrostatically attracted and able to aid PNA uptake through membrane permeabilization (Eriksson et al., 2002). The mechanism of permeabilization is unclear but appears similar to the action of cationic antimicrobial peptides (Eriksson et al., 2002). Peptide-PNAs can provide potent antisense and bactericidal effects in Gram-negative and Grampositive bacteria (Good et al., 2001; Nekhotiaeva et al., 2004). However, improved cell uptake does not ensure favorable effects against infections in vivo. Therefore, there is still much progress needed to improve the efficacy and reduce human toxicity of bacterial cell permeable peptide-PNAs.

B. PNA effects on RNA In principle, all cellular RNAs are potential targets for recognition and inhibition with PNA. As an RNA inhibitor, PNA has several favorable properties. In particular, PNA typically shows high-affinity binding to complementary nucleic acids (Giesen et al., 1998), is stabile against cellular nucleases (Demidov et al., 1994), and has a constrained backbone flexibility that allows binding to structured target RNAs (Dias et al., 2002). Nevertheless, PNA-mediated effects on specific target RNAs can be difficult to achieve, and it is important to consider the properties of PNA and the nature of the RNA target, as discussed in the following passages. The primary mechanism of action for PNA on RNA is almost certainly steric hindrance of translation. There are no reports of PNA–RNA duplexes being recognized or processed by cellular nucleases, and the unnatural PNA backbone is unlikely to be compatible with cleavage by RNase H or RISC complex components. Despite this lack of compatibility with cellular nucleases, PNA can affect RNA in a variety of ways. Steric hindrance of RNA can have many important and useful effects on RNA. Peptide nucleic acids and other antisense agents have been designed to target most of the major classes of RNA in cells to affect gene expression, RNA processing, and RNP activities. Also, PNA binding can potentially alter RNA abundance by triggering nonspecific degradation of unused or structurally altered RNAs. Finally, PNA-based artificial ribonucleases are being developed to enter cells and cleave target RNAs through attached RNA-hydrolyzing moieties (Petersen et al., 2004b). In the subsections given later, we describe PNA recognition of the major classes of RNA and the various biological effects possible in cells and animals.

1. PNA recognition of RNA Cellular uptake is perhaps the most significant barrier against PNA recognition of cellular RNAs. Yet, even where efficient cell uptake can be achieved, RNAs comprise a complex set of potential targets. There are several intracellular

20

Lundin et al.

factors that influence PNA effects on RNA, and each target RNA presents a significant new challenge.

a. Peptide nucleic acid and RNA localization For efficient recognition, localization of the PNA must coincide with the target RNA. Colocalization is a difficult issue because little is known about PNA localization in cells. Fluorescence microscopy studies suggest that PNAs tend to accumulate in endocytic compartment (Folini et al., 2003; Gray et al., 1997). However, there are several reports of nuclear and cytoplasmic RNA inhibition using free PNA (see in a later section), and accumulation in nuclei has been observed (Bonham et al., 1995; Folini et al., 2003). Also, improved endosome escape and antisense potency was observed using endosome disruption strategies (Folini et al., 2003).

b. Higher order RNA structures Secondary and tertiary structures of RNA and their interaction with cellular protein are extremely complicated subjects, and their influence on PNA recognition difficult to predict or assess. However, many RNAs within ribonucleoprotein complexes are attractive as potential drug targets, and PNA is of special interest in this respect, as its flexible backbone allows it to adapt to the structure of complementary sequences (Beck, 2002; Brown et al., 1994).

c. Binding affinity and sequence selectivity Peptide nucleic acid chemistry is expected to provide improved sequence affinity and selectivity at many, but not all, target RNA sequences (Egholm et al., 1993). PNA–RNA hybrid stability can be estimated using algorithms that calculate melting temperature (Tm) values (Giesen et al., 1998), and target site uniqueness can be assessed using bioinformatics sequence analyses tools (Saetrom, 2004). However, predictions made with such tools should be supplemented with comparative studies.

2. Effects on messenger RNA Antisense and RNAi mechanisms have attracted great interest as selective messenger RNA inhibitors (Scherer and Rossi, 2003). To reduce messenger RNA expression using PNA, it is important to target sites where it can block translation initiation. In this regard, assembled ribosomes are very processive, and even tight-binding PNA molecules are unlikely to block elongation (Knudsen and Nielsen, 1996). Successful antisense PNAs typically bind sites within the translation initiation region, where ribosomal components and translation factors assemble prior to elongation (Doyle et al., 2001). An interesting

1. Biological and Biotechnological Aspects of PNA

21

exception to this rule involves triplex forming PNAs, which form extremely stable complexes at purine rich sequences. Also, the entry site for the ribosomal small subunit is a sensitive target for PNA inhibition (Doyle et al., 2001). Despite these guidelines related to susceptibility, a PNA cannot always be designed directly from the mRNA sequence and show effective inhibition of gene expression. Rather, a series of PNA oligomers will usually need to be synthesized and tested. Nevertheless, many successful antisense PNAs have been reported using cell-culture systems, including PNAs targeted against the mdm2 oncogene (Shiraishi and Nielsen, 2004), IL-5R, (Karras et al., 2001), and isoform-selective inhibition of caveolin-1 (Liu et al., 2004). Several reports using mice show that antisense PNAs can inhibit mRNAs. For example, the CXCR3 chemokine receptor, which is believed to play a critical role in the allograft rejection, was significantly inhibited by using intravenous administration of anti-CXCR3-PNA, and this effect marginally prolonged skin allograft survival (Jiankuo et al., 2003). Local injection into the rat brain of a PNA-cell pernetrating peptide conjugate inhibited the galanin receptor expression and activity (Pooga et al., 1998). Also, the amyloid precursor protein (APP) in the brain of mice was reduced up to 70% by an anti-APP PNA (Boules et al., 2004). Finally, in a mouse model of familial ALS, PNA-mediated inhibition of glutamate receptor mRNA and delayed disease onset was demonstrated after repeated intraperitoneal administration (Rembach et al., 2004). Peptide nucleic acid inhibition of bacterial mRNAs may present a more straightforward design challenge. In bacteria, assembly of the translation machinery occurs at the start codon region, and this involves direct recognition between the small ribosomal subunit and the Shine-Dalgarno signal 6–13 bases upstream of the start codon. It is clear from mRNA target site scanning experiments of several genes in bacteria that the start codon region is susceptible to steric hindrance by PNA (Dryselius et al., 2003). Also, improved uptake using carrier peptides is needed for efficient inhibition in wild type Gram-negative and Gram-positive bacteria (Good et al., 2001). Such peptide-PNAs can inhibit bacterial growth when targeting growth-essential genes in Escherichia coli and Staphylococcus aureus (Good et al., 2001; Nekhotiaeva et al., 2004). Similar results and design restrictions have been reported using morpholino antisense oligonucleotides. Reports demonstrate the ability of peptide-PNAs and similar peptide-phosphodiamidate morpholino conjugates to inhibit E. coli bacterial growth and prevent fatal intraperitoneal infection in mice (Geller et al., 2005; Tan et al., 2005). In addition, PNAs are not effectively exported from the bacteria by the efflux pumps that normally eliminate antibiotics such as penicillins (Good et al., 2000). Finally, PNA inhibition at the RNA-level sensitizes bacteria to protein-level inhibitors of related targets (Dryselius et al., 2005). Therefore, there are unique opportunities to develop bactericidal antisense agents based on PNA.

22

Lundin et al.

3. Effects on mRNA splice site selection Following transcription, most cellular RNAs are rapidly processed into their mature form by trans-acting factors that recognize splice-signal motifs within newly transcribed mRNA. It has been estimated that alternative splicing is involved in the expression of >50% of human mRNA (Sharp, 2005). Therefore, exon shuffling dramatically expands the variety of gene transcripts and functional open reading frames. Sequence alterations at the DNA level, are largely unsuited to inhibit or redirect splicing to specific RNA isoforms. Thus, there is great interest in developing antisense agents to inhibit mRNA splice site selection, which could alter the profile of mRNA splice variants in cells. For at least three reasons mRNA splicing reactions may be particularly good targets for PNA. First, PNA works through steric hindrance, which is the most straightforward approach to redirect splicing. Second, PNA can reach the nucleus (Bonham et al., 1995), where splicing occurs. Finally, modest redirection of splice variant levels can cause large biological effects through the increase or reduction of functionally dominant variants. Building on earlier studies using 20 -O-methoxyethyl (20 -O-MOE) antisense oligomers, PNA oligomers were targeted to a splice site that leads to a mature mRNA coding for a membrane-bound form of the interleukin-5 receptor (Karras et al., 2000). Peptide nucleic acid was successfully delivered into cells using electroporation and increased levels of an alternative, soluble form of the receptor. A study by Sazani et al. (2002) took the next step by analyzing antisense PNAs in vivo. An 18-mer PNA with four lysine residues at the C-terminus blocked splicing at a specific site by up to 40% in a range of mouse tissues, following intraperitoneal administration. In most tissues examined, PNA outperformed 20 -O-MOE-phosphorothioate and morpholino analogs of the same sequence.

4. Inhibition of viral RNAs Many medically important viruses contain RNA genomes (Moya et al., 2004), presenting obvious targets for antiviral agents. Viral genomic RNAs contain conserved RNA structures that are essential for viral growth. Also, DNA viruses typically use internal ribosomal entry sites (IRES) to initiate mRNA expression. Therefore, viral RNA appears to present susceptible targets for recognition and steric hindrance. Soon after the discovery of PNA, it was shown that it can inhibit elongation of viral reverse transcriptases on RNA templates (Hanvey et al., 1992; Koppelhus et al., 1997). By focusing on well defined, conserved, and essential viral RNA structures, several laboratories have shown that PNAs can inhibit viral replication in vitro and in infected cells. For example, PNAs have been designed to target sequences needed for RNA genome dimerization and

1. Biological and Biotechnological Aspects of PNA

23

transcription, including the TAR hairpin loop (Chaubey et al., 2005; Mayhood et al., 2000; Riguet et al., 2004; Tripathi et al., 2005), and the HIV Rev-response element (RRE), which binds to viral peptides at the same RNA site (Kumagai et al., 2001). Also, experimental antiviral PNAs have been developed to target an IRES element in the Hepatitis C Virus (HCV) (Nulf and Corey, 2004), and HBV has been inhibited in duck primary hepatocytes using PNA (Robaczewska et al., 2005).

5. Inhibition of ribonucleoprotein complexes a. Ribosomes Ribosomal RNA is the most abundant form of cellular RNA. The RNA component makes up approximately 40% of the total mass of a ribosome and plays an active role in translation. While most rRNA is inaccessible to sequence recognition, the active regions are often exposed (Saxena and Ackerman, 1990). Many useful antibiotics target ribosomal RNA, and these sequences are also accessible to hybridization (Cundliffe, 1987). Active regions of rRNA tend to be highly conserved; however, there may be sufficient divergence within essential regions to allow bacteria specific anti-rRNA PNAs. As a first step, PNAs were targeted to two regions of ribosomal RNA, the alpha-sarcin loop and the peptidyl transferase center, and found to inhibit translation in vitro and bacterial growth (Good and Nielsen, 1998). Also, potency was improved by using attached carrier peptides. Therefore, it may be possible to develop PNA-based translation inhibitors as effective antimicrobials.

b. Telomerase Telomerase is a ribonucleoprotein that maintains telomere length. In most tumor cells, telomere length expansion appears to prevent senescence and growth arrest, while elevated telomerase activity is rarely observed in normal somatic cells (Autexier, 1999). Repeating sequences of human telomeres (50 -TTAGGG-30 ) are synthesized by the reverse transcriptase activity of telomerase. Repeat extension is accomplished using a complementary and accessible RNA template that is also part of telomerase. The RNA that provides this template sequence is an accessible target for PNA, and several studies show that PNAs can inhibit telomere extension in vitro with low-nanomolar IC50 values. This compares well with analogous phosphorothioate oligomers and 20 -O-methyl RNA oligomers, but in cell-culture experiments 20 -O-methyl RNA oligomers appear more effective (Hamilton et al., 1997). However, antitelomerase PNAs were only effective if present before assembly of the full RNA–protein complex. If the PNAs were added after telomerase assembly, they exhibited virtually no activity, suggesting that access to the target sequences may be blocked by the tertiary structure of the RNA–protein complex.

24

Lundin et al.

Therefore, only selected PNAs and their careful application have shown antitelomerase activity in cell culture.

C. PNA in gene regulation The capacity of PNA to strand-invade into dsDNA has also opened the possibility of interfering directly with gene regulation. Provided that the PNA is transported all the way into the cell nucleus, (see in an earlier section), PNA may act either as a transcription activator or inhibitor (“antigene”). Both possibilities have been covered in several reviews (Marin et al., 2004; Nielsen, 2005). To affect gene expression at the DNA level, it is important to consider the capacity for PNA to strand-invade into dsDNA, in the context of chromatin under physiological conditions. In addition, interference with gene expression by acting as a decoy molecule binding different transcription factors has been addressed.

1. Using PNA to increase specific gene transcription a. Transcription initiation from PNA-displaced ssDNA loops When PNA binds to dsDNA via strand displacement, a single-stranded DNA loop (D-loop) is formed. The possibility that this displaced DNA strand could facilitate transcription of genes downstream of the PNA-binding site has been investigated both in vitro and in vivo (Mollegaard et al., 1994; Wang et al., 1999, 2001). The E. coli RNA polymerase displays high affinity for ssDNA and is able to initiate transcription in vitro from short (12 bp) mismatched stretches within a dsDNA template (Aiyar et al., 1994). Mollegaard et al. (1994) reported that the D-loop generated by a 10-bases long pyrimidine-PNA was also enough to permit initiation of the transcription by a T7 RNA polymerase in vitro; the activity was comparable with that achieved by the lacUV5 promoter. In addition, transcription was also PNA-induced in a rat nuclear extract, indicating that transcription initiation can occur with eukaryote RNA polymerases as well. Two short homopyrimidine PNAs, 10 and 12 bases long, were later reported to induce transcription of the green fluorescence protein (GFP) reporter gene in vitro in HeLa-cell nuclear extract as well as in vivo in CV1 monkey kidney fibroblast cells after microinjection of preformed plasmid-PNA hybridization complexes (Wang et al., 1999). In contrast to what was reported for the E. coli polymerase, which initiated transcription from several positions within the ssDNA loop, in the nuclear extract transcription was shown to start at the 30 end of the D-loop. Not unexpected, stronger activity was generated from the D-loop caused by the longer PNA. The optimal length of a PNA, to act as artificial promoter, was later determined (Wang et al., 2001). Peptide nucleic

1. Biological and Biotechnological Aspects of PNA

25

acids 14–20 bases long were shown to induce transcription in vitro in HeLacell nuclear extract, with the strongest activity induced by the 18-mer. Also, PNAs induced expression from a promoterless GFP-reporter plasmid in cell culture. Again, the strongest signal was achieved with 16–18 bases long PNAs. In this experiment, the transfection of hybridized plasmid was performed using cationic liposomes (Wang et al., 2001). These findings have, however, so far been difficult to reproduce in other laboratories (Nielsen, 2005) and our own unpublished results. Wang et al. (1999) also reported increased transcription of the -globin gene in the human erythroleukemia cell line K562 after introduction of PNA via electroporation. The normally very low-endogenous expression of -globin mRNA increased 2.8-fold after treatment with PNA designed to bind to the 280 region of the gene. Analysis of transcripts showed that initiation was not only occurring at the PNA-binding site, but also that increased transcription was achieved from the natural promoter (Wang et al., 1999). Later it was found that the 280 region is important for binding the transcription factor Oct-1, which is known to repress the transcription from the -globin gene. By targeted mutagenesis within this region reduced Oct-1 binding was achieved, resulting in increased gene expression in MEL cells (Xu et al., 2000). Accordingly, PNA binding to this region might reduce the Oct-1 binding due to steric hindrance, allowing for increased transcription from the original promoter.

b. Transcription initiation by a PNA-anchored artificial activation domain Transcription activation by a PNA-peptide chimera in vitro in a mammalian nuclear extract has also been reported (Liu et al., 2003). The peptide part was earlier selected to bind to the Gal80 transcriptional repressor and shown to mimic the Gal4 activation domain in yeast. The double-stranded template DNA contained PNA-binding sites positioned in the promoter proximal region, upstream of a TATA-box and a G-less reporter sequence. Peptide nucleic acid was hybridized at high-molar excess and the DNA–complex purified prior to incubation in the HeLa-cell nuclear extract. Bound PNA without peptide reduced the original transcription almost completely from the DNA fragment containing five binding sites. The effect was less pronounced using a fragment with a single binding site. Hybridization with the PNA-Gal80-binding peptide, however, completely restored transcription. For the fragment containing five binding sites, transcription was slightly higher in the presence of the PNA-peptide than without PNA. No effects were seen when using fragments missing the PNA-binding sites. In addition, these authors discussed their lack of success when trying to induce transcription of a reporter gene in vivo in cell cultures. In these experiments the DNA was prehybridized with the PNAGal80-binding peptide prior to transfection. The lack of positive expression

26

Lundin et al.

was speculated to be due to intracellular degradation of the peptide (Liu et al., 2003). Notably, though, sensitivity in the in vitro experiments was much higher with a read-out measurement of 32P-labeled RNA-fragments, as compared with detection of protein production from a reporter gene.

2. Using PNA to block gene transcription Antigene effects of PNA have been reported from in vitro transcription experiments, as well as in cell cultures and in live animal studies. In the early 1990s it was found that PNA2/DNA complexes formed on the template strand at sites within or downstream of the promoter could block transcription elongation in vitro (Hanvey et al., 1992; Nielsen et al., 1993a, 1994). The construction of bisPNA and the introduction of J bases increased the possibility for PNA strand invasion under physiological conditions (Egholm et al., 1995). Later, it was described that active transcription of the DNA facilitated binding of PNA not only to the sense but also to the template strand, due to the opening up of the dsDNA by the moving polymerase (Larsen and Nielsen, 1996). The construction of “tail-clamp” PNAs consisting of a short bisPNA combined with a mixedbase PNA tail has reduced the requirement for long purine stretches as target sequence for efficient strand invasion (Bentin et al., 2003; Kaihatsu et al., 2003). Such “tail-clamp” PNAs can efficiently block transcription in vitro, further adding to the possibility of using PNA as antigene molecules to turn off transcription. While using PNA to induce artificial transcription in vitro in mammalian nuclear cell extracts, Liu et al. (2003) also found that PNA without activation peptide could block RNA polymerases, despite the fact that it was binding to the coding DNA strand. Notably, the presence of multiple PNA-binding sites resulted in a more pronounced inhibition. Others have found that it is possible to block specific protein expression by simply adding “antigene” PNAs into cell cultures. In several studies, positively charged NLS peptides were used to facilitate cellular as well as nuclear uptake of PNA (Boffa et al., 2000; Cutrona et al., 2000). It was also reported that a 15-mer PNA-NLS chimera, complementary to a sequence found once within the pol gene and once within the nef gene of HIV-1, both positions being conserved within the viral populations, blocked viral production in infected cells (Pesce et al., 2005). A concentration of 10 mM PNA significantly reduced HIV replication both in lymphocytes and in chronically infected macrophages. Whether the PNAs actually act via strand invasion was, however, not investigated in any of these reports. In actively transcribed genes, DNA is less densely arranged within the chromatin, due to the action of acetylases and other protein complexes ( Jenuwein and Allis, 2001). Together with the dynamic breathing of the negatively supercoiled DNA (Bentin and Nielsen, 1996) the transient opening

1. Biological and Biotechnological Aspects of PNA

27

up of the DNA strands by RNA polymerases further facilitates PNA strand invasion. This may partly explain why a linear mixed-base PNA, which does not bind to linear dsDNA by strand displacement in vitro, still gives clear effects on gene expression in vivo. Kaihatsu et al. (2003) also found reduced in vitro transcription with both linear and tail-clamp PNAs peptide conjugates that were not able to display strand invasion, as analyzed by mobility shifts in gel electrophoreses of a linear dsDNA fragment containing the specific PNAbinding site. In a later report, Cutrona et al. (2003) showed that specific downregulation of c-myc transcription could be achieved with a PNA complementary to a noncoding region upstream of the translocated oncogene. In some Burkitt’s lymphoma cell lines the translocated c-myc is positioned just downstream of the Em enhancer region on chromosome 14, while in other cell lines the chromosomal break occur upstream of the Em enhancer, which is then translocated to chromosome 8. In this study, the Em-specific PNA only induced downregulation of the c-myc mRNA and protein production in the Burkitt’s lymphoma cell line where the Em enhancer was present just upstream of the c-myc gene. In a cell line where the Em enhancer was translocated together with the heavy chain gene, the same PNA only blocked the production of the heavy chain (Cutrona et al., 2003). This study provides a beautiful set up for excluding other unspecific intracellular mechanisms for the action of PNA, and blocking studies proved specific binding of PNA to dsDNA fragments containing the correct sequence. Whether the mechanism involves true strand displacement was, however, not demonstrated (Cutrona et al., 2003). Mixed linear PNAs require supercoiled DNA for specific strand displacement in dsDNA and are rapidly “kicked out” by the competing strand when the DNA relaxes or the ion strength is increased (Lundin et al., 2005). Larger PNA complexes appear, however, to build up at the specific PNAs binding sites, allowing for steric hindrance of different proteins normally acting on the DNA target. In our hands, bisPNA prehybridized to plasmids prior to transfection seemed to require the build up of large complexes in order to block gene expression in vivo in cell culture. Prehybridized plasmid, from which excess PNA was removed before transfection, did not induce blocking of intracellular transcription, despite 100% hybridization, as verified by gelshift analysis (Ge et al., manuscript). Binding of PNA-complexes to DNA is not only capable of arresting DNA polymerase elongation but has also been shown to block DNA helicases from unwinding dsDNA-substrates in vitro (Bastide et al., 1999). Injection into rats of unmodified mixed-bases PNA, has been reported to specifically block gene transcription in vivo. Direct injection into the brain was reported to downregulate neurotensin receptor-1 mRNA transcription, leading to reduced protein expression (Tyler et al., 1998). Also intraperitoneal injection of rats with an unmodified PNA specific for the angiotensinogen gene

28

Lundin et al.

resulted in reduced blood pressure and downregulation of the specific mRNA, without detectable reduction of mRNA for four different control proteins (McMahon et al., 2002b). The biological effects of the administration of very short unmodified mixed-bases PNAs are very interesting, since strand-invasion into dsDNA under physiological conditions has never been shown to occur in vitro. Active transcription of the gene, as discussed in an earlier section, might be one possible explanation. Further studies to investigate the general capacity of unmodified as well as modified PNAs to pass over the cell membrane, as well as over the blood–brain barrier, are also of highest interest for the development of PNAs as gene-modifying drugs.

3. PNA containing “decoy” transcription factor responsive elements Transcription factors typically recognize double-stranded consensus sequences within gene promoter regions. Decoy molecules are short double-stranded nucleic acids designed to mimic genomic transcription factor-binding sites and sequester factors that may be overactive in disease. The transcription factor NF-B recognizes a well-characterized responsive element, and this factor is activated in many forms of cancer and inflammation (Pande and Ramos, 2005). Short DNA duplexes containing the NF-B responsive element act as efficient decoys of NF-B in vitro and show anti-inflammatory activities in animal models of inflammation (Morishita et al., 2004). Several studies have investigated the use of nucleic acid analog incorporation within decoy sequences to improve drug properties, with significant progress reported. As expected, PNA–DNA decoy structures show improved nuclease resistance (Borgatti et al., 2003b); but, somewhat surprisingly, PNA–DNA hybrids containing a responsive element are recognized by NF-B (Mischiati et al., 1999). PNA–DNA decoys have shown potent NF-B inhibitory effects in cells (Fisher et al., 2004; Penolazzi et al., 2004). Also, PNA–DNA chimeric molecules inhibit the Sp1 transcription factor both in vitro and in cell culture (Borgatti et al., 2003a). Therefore, nuclease-stable PNA–DNA and PNA–DNA chimera decoys may be recognized by transcription factors and provide new possibilities to improve decoy technology aimed at controlling aberrant transcription factor activity.

D. Future prospects for PNAs as antisense and antigene drugs For research applications, PNA is used to inhibit and redirect the activities of mRNA, viral RNAs and RNPs; and these important RNAs will remain popular targets. Peptide nucleic acids may also prove useful as tools to study other types of structural RNAs and the wide variety of previously unknown small noncoding RNAs, which have been discovered, including large numbers of short and long

1. Biological and Biotechnological Aspects of PNA

29

natural antisense transcripts. In addition, PNA promises to be a useful tool to study molecules and mechanisms involved in gene transcription, for example, RNA polymerase transcription termination (Guffanti et al., 2004). For possible clinical applications, PNA compares well with other antisense technologies, whereas more studies involving comparison to RNAi are needed. Increasing in vivo data also suggest that PNA may be a possible antigene candidate, although the mechanism of action requires further investigation. While PNA lacks the ability to recruit cellular nucleases as its primary mechanism of action, it is important to consider that steric hindrance is the mechanism of action of most successful drugs. Also, the lack of specific recognition by cellular proteins suggests that PNA effects are unlikely to be complicated by stimulation of toll-like receptors (TLRs), which signal immune responses following binding with certain DNA motifs and double-stranded RNA (Sivori et al., 2004). Finally, PNA pharmacokinetics is acceptable for drug development, and biodistribution can be altered through modification (Hamzavi et al., 2003b). Therefore, the long-term prospects for PNA as antisense or antigene drugs in vivo are promising.

IV. BIOTECHNOLOGICAL ASPECTS OF PNA The biotechnological aspects of PNA cover not only biomedical applications and diagnostic use but also more basic uses of PNA in molecular biology. The possibility of using PNA in the field of nanotechnology as tools for assembling macromolecules in microchips, and so on will, however, not be included in this chapter.

A. PNA-anchors acting as genetic “glue” Due to the high-binding affinity between PNA and natural nucleic acids, both mixed-bases “linear” PNA and pure pyrimidine containing bisPNA clamps have been used to attach molecules to DNA in a sequence-specific manner. Mixed-base PNAs are mostly used as anchors for binding fluorophores, peptides, or carbohydrates to single-stranded nucleic acid molecules. Such PNAs can only perform strand invasion into dsDNA under certain conditions—the target must be supercoiled, and the hybridization should occur at low-ion concentrations. Bound PNA will rapidly dissociate at physiological salt concentrations even when the competing DNA strand is partly occupied by additional PNAs (Lundin et al., 2005). Only where both strands are displaced, “linear” mixedbases PNA-anchors will remain stably hybridized under physiological conditions. This can be achieved using so-called pseudo-complementary PNAs, where the use of modified base analogs prevent PNA–PNA hybridization (see Fig. 1.3)

30

Lundin et al.

(Demidov et al., 2002; Lohse et al., 1999). For binding to double-stranded DNA, bisPNA clamps provide higher stability (Egholm et al., 1995). The restriction in target sequence composition and the requirement for longer PNA-molecules (two PNA-bases per base in the target sequence) is the price paid for the enhanced complex-stability. The idea of “gluing” biological functions to nucleic acids in a sequencespecific manner has been particularly attractive in the field of gene therapy, in order to improve the efficiency of nonviral gene transfer. Short peptide sequences known to mediate active transport of proteins into the cell nucleus, so-called NLS, have also been used to increase the translocation of DNA across the nuclear membrane (Munkonge et al., 2003). Coupling methods based on chemical linkage of NLS-peptides to plasmid DNA, however, reduce gene expression due to random binding. Most NLS peptides contain several positively charged amino acids (viz., lysines and arginines), and unspecific charge-interactions have also been used to bind such peptides to DNA. These interactions create relatively unstable and poorly defined complexes. The use of large amounts of highly charged peptides also causes extensive DNA condensation, which can result in reduced gene expression due to inefficient decondensation inside the nucleus. Brande´n et al. (1999) were the first to report the use of PNA as anchors to link the SV40 NLS-peptide to a plasmid via sequence-specific hybridization. The trans-gene expression was shown to increase up to eight times after polyethyleneimine-mediated transfection of cells with plasmids that had been prehybridized with a mixed-base PNA-NLS-peptide-chimera. The method to form transfection complexes containing biologically active entities linked to DNA via PNA-anchors (Fig. 1.6) was later designated

Figure 1.6. Schematic illustration of the Bioplex technology. Functional entities are bound via flexible hydrophobic linkers to PNA-anchors, which then are hybridized to DNA in a sequence-specific manner. The different functions may be linked in single or multiple molecule manners, by the use of branched linkers.

1. Biological and Biotechnological Aspects of PNA

31

“Bioplex” formation (Brande´n and Smith, 2002; Svahn et al., 2004a). Biotinlabeled bisPNAs-binding streptavidin-conjugated molecules, and maleimideactivated PNAs, which can bind cystein-containing peptides, have been used to attach functional peptides in a similar fashion (Zelphati et al., 2000). Bremner et al. (2004) also used maleimide-activated bisPNA prehybridized to plasmids to anchor four different NLS-peptides to DNA, in order to compare the effects of the different NLS-peptides on transfection efficiency. They used a plasmid containing 5 PNA-binding sites and found that, on average, only 1.6 copies of the NLS-peptides were attached per plasmid despite efficient (80%) PNA binding. No significant increase in transgene expression could be detected for three of the four peptides tested, including the short SV40 peptide. This was likely due to the very low number of peptides binding to the PNA when already hybridized to the plasmid. According to Brande´n et al. (1999) plasmids with 11 (but not 2) binding sites were shown to give enhanced transgene expression. To achieve a well defined complex it is, however, safer to synthesize the PNA as a peptide chimera or to attach the functional entity by conjugation prior to plasmid hybridization. The risk for steric interference between functional groups on adjacent PNA-anchors is then easily investigated, and the distance between the binding sites can be optimized. By analyzing binding kinetics of linear PNApeptides, the optimal distance between the binding sites on the same DNAstrand was found to be two bases (Lundin et al., 2004). Steric interference as well as charge may be important for binding efficiency during “Bioplex” formulation. The use of “opener-assisted” bisPNA hybridization, using another nucleic acid analog, called locked nucleic acid (LNA), as an “opener” molecule, has been reported to reduce both the time and amount of bisPNA needed to achieve more than 90% hybridization of multisite plasmids (Lundin et al., 2005). NLS-containing “Bioplex” has also been shown to induce increased nuclear translocation of fluorophore labeled oligonucleotides after subcutaneous injection in mice (Brande´n et al., 2001). Ligand binding to specific cell-surface receptors confers the possibility to introduce cell-specific uptake of transfection complexes. Two attractive molecules are the carbohydrate N-acetyl-galactosamine (GalNac) ligand for the ascialoglycoprotein receptor (Wu et al., 2002) and the RGD peptide ligand for the v3 integrin receptors (Wittekindt et al., 2004). Transferrin-conjugated PNA has also been used and, when hybridized to plasmids, was shown to induce a fourfold increase in polyplex-mediated transfection of myoblasts (Liang et al., 2000). So far, cell-specific uptake of plasmids hybridized with PNA-peptide/ conjugates alone, without assistance of other transfection methods, has not been reported. To achieve this, a mechanism to induce endosomal escape is required, and the plasmid likely must be condensed in order to reduce the size of the complex and protect the DNA. For a review of the intracellular trafficking of

32

Lundin et al.

cellular receptors see Gaborik and Hunyady (2004). The steroid hormone dexametasone has also been attached to plasmids via a bisPNA-anchor and was shown to give a 20-fold increase in reporter gene expression after lipoplex-mediated transfection of nondividing cells (Rebuffat et al., 2002). Many hormone receptors, such as the glucocorticoide receptor, are localized in the cytoplasm and, after binding the ligand, actively translocated into the cell nucleus. This strategy, to facilitate the intracellular transport of introduced DNA, has been termed steroid-mediated gene delivery (SMGD); the method is described in detail by Rebufat et al. (2004). Thus, choosing different hormones as functional entities may further increase the tissue specificity for targeted gene delivery. bisPNA and LNA, which also displays a very high affinity and specificity for complementary nucleic acid sequences, have been used to attach oligonucleotides containing the CpG motif to plasmids in a sequence-specific manner (Hertoghs et al., 2003). The CpG motif is known to increase the immunostimmulatory effect of bacterial DNA, acting via Toll-like receptor 9 (Bauer et al., 2001; Hemmi et al., 2000). Oligonucleotides containing such sequences might act as an adjuvant to increase the immune response after DNA vaccination, provided that binding to the protein-expressing plasmid is sufficiently stable. Hertoghs et al. (2003) reported that LNA but not bisPNA-anchored CpGcontaining oligonucleotides remained attached to the plasmid under high-salt conditions (1 M CaCl2) used to coat DNA on gold particles for gene-gun delivery. Thus, under extremely high-salt concentrations, the use of PNA to bind labels or other molecules providing specific functions to the DNA may not be the optimal method, but under physiological conditions the binding has proved to be exceptionally stable.

B. PNA beacons and other fluorescent PNA-probes Due to its high affinity and sequence specificity, and the fact that the PNA hybridization to DNA and RNA is relatively insensitive to salt concentrations, PNA has become a useful tool as genetic probe. For example, increased fluorescence signals after nucleic-acid hybridization has been used to detect the presence of a specific DNA sequence after PCR amplification and in genomic DNA samples as well (as discussed in a later section).

1. PNA beacons Molecular beacons are probes that contain a fluorophore and a quencher group attached at opposite ends of the oligonucleotide (Tyagi and Kramer, 1996). As long as the probe remains unhybridized the ends are close enough to induce

1. Biological and Biotechnological Aspects of PNA

33

quenching of the signal from the fluorophore. While binding to the target, the ends become separated, and the fluorescence is regained (Fig 1.7A). The first reported use of PNA to detect PCR products used a chimeric DNA–PNA probe (Ortiz et al., 1998), but the method was rapidly improved to involve pure PNA probes (Seitz, 2000). In PNA beacons, the hairpin structure originally used for DNA beacons is not required. The hydrophobic structure and lack of charge repulsions in the PNA is sufficient to favor a condensed structure with quenched signals from the free PNA probe. Peptide nucleic acid probes are also less dependent on salt concentrations, and less sensitive to the presence of proteins that bind to ssDNA. This allows detection of nucleic acids directly in different buffers and protein-containing solutions (Kuhn et al., 2001, 2002; Seitz, 2000). Because PNA-beacon molecules are made as “linear” PNA, the probes require assistance of “openers” to enable access and recognition of sequences in dsDNA. Binding of bisPNA to oligopurine sites flanking the target site creates extended strand-displacement complexes, allowing the binding of the probe to the displaced strand (Kuhn et al., 2001, 2002). According to Bukanov et al. (1998), two short and closely spaced oligopurine sites should statistically occur at, on average, every 400–500 bases in a random DNA sequence. Peptide nucleic acid molecular beacons have also been used to detect single nucleotide polymorphism (SNP) via real-time PCR. Petersen et al. (2004a) used a PNA molecular beacon probe to detect SNP in exon 6 of the XPD gene, coding for a protein involved in DNA repair and for which the mutation in question correlates with enhanced risk for basal-cell carcinoma. They found that the PNA probe gave a better discrimination between the two alleles. However, the signal from the PNA-beacon was lower than the signal achieved for the standard TaqMan analysis used in comparison.

2. Light-up probes An alternative method for rapid detection of PCR-amplified DNA using a PNA labeled with the fluorophore thiazole orange (TO) has been described (Svanvik et al., 2000; Wolffs et al., 2001). While bound to DNA, the fluorescence from TO increases significantly (Fig 1.7B), a property that remains after conjugation to PNA. However, the background fluorescence of unbound PNA-TO probes varies depending on the sequence of the PNA probe (Svanvik et al., 2001). This “light-up” probe technology has been further developed by introduction of forced-intercalation probes (FIT-probes) (Fig 1.7C). They show strong fluorescence after matched hybridization, while the fluorescence becomes attenuated following mismatched hybridization (Kohler et al., 2005). The “light-up” probe technology is now applied in clinical diagnostic tests and has also been used to investigate the binding kinetics of PNA-peptides binding to super-coiled plasmids (Lundin et al., 2004).

34

Lundin et al.

Figure 1.7. Schematic representations of different fluorescent PNA probes. (A) Molecular beacons, in with the fluorescent group is quenched when the probe is present in solution and free to emit light after hybridization to DNA. (B) The light-up probe, which is weakly fluorescent when in solution and strongly fluorescent when the dye is allowed to intercalate into the PNA–DNA duplex. (C) The FIT fluorescent probe where the intercalating dye serves as a base surrogate and emits light only after matched hybridization.

1. Biological and Biotechnological Aspects of PNA

35

3. Cyanine dye staining of PNA–DNA duplexes A third method to detect the formation of PNA–DNA duplexes is to stain with the cyanine dye DiSc2(5). This dye binds with high affinity to PNA–DNA duplexes, which induces a color change from blue to purple (Komiyama et al., 2003; Wilhelmsson et al., 2002). Using an anionic form of the cyanine dye a modified assay to detect DNA–PNA interaction by increased fluorescence was presented (Datta et al., 2004). Unfortunately, the use of both the colorimetric and the fluorescence-based methods are limited, due to a highbackground signal from both ss- and dsDNA.

4. PNA in fluorescence in situ hybridization The capacity of PNA to hybridize with high affinity to DNA under low-ionic conditions allows binding of PNA probes to denatured DNA samples under conditions where the reannealing of separated DNA strands is less favorable. Together with the remarkably high stability toward enzymatic degradation this has made PNA useful both to provide specific probes and to block repetitive sequences in different FISH-protocols. For recent reviews, see (Nielsen et al., 2004; Pellestor et al., 2005). The possibility to use short PNA probes has also allowed the development of sequential staining and repeated analysis of different chromosomes within the same cell (Paulasova et al., 2004). Sequential FISH with probes specific for eight different chromosomes was reported for analyzing blastomers in preimplantation genetic diagnostics (Agerholm et al., 2005). Methods using PNA-FISH targeting rRNA has also been established for rapid typing of both bacteria and yeast (Stender, 2003).

C. Other biotechnological applications

1. PCR clamping Small differences between a given set of DNA or RNA sequences can be detected by PCR/RT-PCR, either by specific amplification of the mutated and not the wild-type sequence or by preventing the amplification of the mutated fragment. To increase sensitivity for detecting single-base differences in target sequences, bisPNA-clamps have been used in reactions known as “PCR Clamping.” Using a PNA specific to the original sequence, amplification of the wildtype fragment can be blocked. Due to extremely high-sequence discrimination in PNA–DNA hybridization, a single-base mismatch is enough to prevent PNA binding, and sequences with as little as one base difference can be amplified. By introducing a second annealing step for the PNA clamp at a temperature above the Tm for the primer, effects of reduced PNA concentrations and binding

36

Lundin et al.

kinetics are minimized. The general technique and selected applications of the PCR clamping is nicely reviewed by Ørum (2004). PCR clamping provides a helpful tool when mutation analysis is made on the final PCR products. For realtime PCR detections, the fluorophore-based methods discussed earlier may to be more optional (Kohler et al., 2005; Petersen et al., 2004a).

2. PNA as artificial restriction enzymes It was recognized early that PNA might be used to introduce site-specific digestion of DNA by S1 nuclease, directed to the displaced ssDNA at the PNA-binding site (Demidov et al., 1993). A possibility to create artificial restriction enzymes has long been highly desired, to facilitate manipulation of large genomic DNA molecules, as reviewed in Komiyama and Sumaoka (1998). Single-stranded DNA was site-specifically cleaved by hydrolysis using a PNA conjugated with Zr(IV)-complexes (Zelder et al., 2003). In addition, sequencespecific nicking in dsDNA has been achieved with the help of PNA “openers” (Kuhn et al., 2003). By hybridizing the target DNA with two short bisPNAs, flanking the specific restriction site, together with an oligonucleotide complementary to the displaced DNA strand, a new restriction site was created on the D-loop. Thus digestion with the corresponding enzyme created site-specific nicking in the target DNA. Yamamoto et al. (2004) described site-specific scission of dsDNA with the help of pseudo-complimentary PNA and Ce(IV)/EDTA. The Ce(IV)/ EDTA complex preferentially hydrolyzes the DNA backbone in gap-sites; and by using only partially overlapping pseudo-complimentary PNAs, the hydrolysis results in cuts with “sticky” ends. Successful ligation of fragments from two different preparations confirmed the possibility to use this method. Efficiency in religation after the Ce(IV)/EDTA hydrolysis was, however, not very high; and successful ligation was verified only after PCR amplification of ligated material. Peptide nucleic acid and Ce(IV)/EDTA cleavage worked equally on both linear and super-coiled DNA. S1nuclease digestion of the same DNA– PNA complexes resulted in smeary bands and, with time, total degradation of the target DNA.

3. Blocking methylation and enzyme digestion An alternative way to reduce the number of cutting sites in a stretch of genomic DNA is to modify naturally occurring enzyme cleavage sites. Peptide nucleic acid can efficiently block many types of DNA-binding proteins, including restriction enzymes (Nielsen et al., 1993b) and methylases (Veselkov et al., 1996), provided that the binding sites are overlapping. Frank-Kamenetskii and

1. Biological and Biotechnological Aspects of PNA

37

coworkers made an early proposal for the use of PNA to induce rare cleavage of genomic DNA. By blocking DNA methylation at selected positions only those few sites remained susceptible for the restriction enzyme (Veselkov et al., 1996). Initially only PNAs recognizing homopurine sites were bound stably enough at salt concentrations required for enzyme activation. With the development of pseudo-complimentary PNAs (Lohse et al., 1999) the possibility to target both DNA strands simultaneously allowed mixed-bases PNA to remain stably bound at higher salt concentrations. Consequently, pseudo-complementary PNAs were tested for their capacity to protect DNA from methylation at sites containing mixed purine and pyrimidine bases, as evaluated by remaining susceptibility for enzyme digestion (Izvolsky et al., 2000). Notably, protection against methylation was less complete when the methylation site was placed at the end of the PNA-binding site. A review about and a protocol for PNAassisted rare cleavage (PARC) are found in Frank-Kamenetskii and Demidov (2004).

4. PNA induced targeted mutagenesis The possibility to use triplex-forming oligonucleotides (TFOs) as a tool to induce site-specific mutagenesis has long been highly attractive, and advances using short donor sequences to induce site-directed recombination have raised prospects for achieving specific gene correction (Kuan and Glazer, 2004). Highaffinity binding of TFOs increased site-specific mutation rates in treated cells. Studies of cell lines deficient in proteins involved in DNA repair indicated that excision repair and transcription-coupled repair mechanisms are involved (Wang et al., 1996). Peptide nucleic acid has attracted interest in this field, as well. Faruqi et al. (1998) used a J-base containing bisPNA specific for a site within the supFG1 gene integrated in the genome of transgenic mouse fibroblasts and found a 10-fold increase in mutation rates within this specific gene (Faruqi et al., 1998). Even though the mutation frequency was still quite low, 0.1%, hybridization was performed inside the cells after membrane permeablization with streptolysine-O. Peptide nucleic acid has also been used as anchors for “donor” DNAs used to induce site-directed recombination in vitro in HeLa-cell extracts (Rogers et al., 2002). When bisPNA was targeted to a site close to the recombination site, the correction frequency increased 50-fold above background and 5-fold above that achieved with the donor DNA alone, also without physical coupling between the PNA-anchor and the donor DNA. In addition, bisPNA on its own was shown to increase repair-induced DNA synthesis 16 times above background, as detected in cell extracts by 32P-dCTP incorporation in PNAhybridized plasmids in vitro (Rogers et al., 2002).

38

Lundin et al.

V. CONCLUDING REMARKS New versions of PNA continue to emerge. However, the original design remains popular, as it provides an appropriate balance between the requirements for high-affinity binding and stringent sequence specificity. The possibilities for easy modification, by adding functional peptides or attaching dyes or ions, which will increase solubility, cellular uptake, and confer cellular specificity, should aid the development of PNA into clinically useful molecules and applications. Although PNA displays a very high stability against enzymatic degradation, and the numbers of studies involving effects of PNA and PNA-conjugates in vivo are increasing, extensive pharmacological studies are still missing. The future use of PNA in vitro is much easier to predict. There are numerous areas within both biomedicine and biotechnology where PNA has drastically changed the possibility to detect and identify even single-base differences between nucleic acid molecules, also when the difference is present in a few copies within a large background. In fields as diverse as genetic mutation analyses, cytology, and viral or bacterial infections, PNA has provided improved possibilities for rapid diagnosis. Also within the field of gene therapy, with the possibility to sequence-specifically label DNA with different biological functions, PNA provides a promising tool. These tactics and techniques are vigorously under development.

Acknowledgments This work was supported by Aroseniusfonden, the Swedish Research Council, the Swedish Science Foundation, the Wallenberg Foundation, the Swedish Foundation for Strategic Research BioXgrant, and by the European Union projects EURO GENE DRUG (QLK 3-CT-2002–01997) and Nucleic Acid-Based Nanostructures (NMP4-CT-2004–013775).

REFERENCES Adlerz, L., Soomets, U., Holmlund, L., Viirlaid, S., Langel, U., and Iverfeldt, K. (2003). Downregulation of amyloid precursor protein by peptide nucleic acid oligomer in cultured rat primary neurons and astrocytes. Neurosci. Lett. 336(1), 55–59. Agerholm, I. E., Ziebe, S., Williams, B., Berg, C., Cruger, D. G., Petersen, G. B., and Kolvraa, S. (2005). Sequential FISH analysis using competitive displacement of labelled peptide nucleic acid probes for eight chromosomes in human blastomeres. Hum. Reprod. 20(4), 1072–1077. Epub 2005 Jan 21. Aiyar, S. E., Helmann, J. D., and deHaseth, P. L. (1994). A mismatch bubble in double-stranded DNA suffices to direct precise transcription initiation by Escherichia coli RNA polymerase. J. Biol. Chem. 269(18), 13179–13184. Autexier, C. (1999). Telomerase as a possible target for anticancer therapy. Chem. Biol. 6(11), R299–R303.

1. Biological and Biotechnological Aspects of PNA

39

Barawkar, D. A., and Bruice, T. C. (1999). Deoxynucleic guanidines/PNA (DNG/PNA) chimeras: Oligonucleoside analogue containing cationic guanidinium and neutral amide linkages. J. Am. Chem. Soc. 121(44), 10418–10419. Barawkar, D. A., Kwok, Y., Bruice, T. W., and Bruice, T. C. (2000). Deoxynucleic guanidine/ peptide nucleic acid chimeras: Synthesis, binding and invasion studies with DNA. J. Am. Chem. Soc. 122(22), 5244–5250. Bastide, L., Boehmer, P. E., Villani, G., and Lebleu, B. (1999). Inhibition of a DNA-helicase by peptide nucleic acids. Nucleic Acids Res. 27(2), 551–554. Basu, S., and Wickstrom, E. (1997). Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insulin-like growth factor 1 for increased cellular uptake. Bioconjug. Chem. 8(4), 481–488. Bauer, S., Kirschning, C. J., Hacker, H., Redecke, V., Hausmann, S., Akira, S., Wagner, H., and Lipford, G. B. (2001). Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA 98(16), 9237–9242. Beck, F. (2002). Solid phasesynthesis of PNA oligomers. In “Peptide Nucleic Acids, Methods and Protocols” (P. E. Nielsen, ed.), pp. 29–41. Humana press, Totowa, NJ. Belting, M., Sandgren, S., and Wittrup, A. (2005). Nuclear delivery of macromolecules: Barriers and carriers. Adv. Drug Deliv. Rev. 57(4), 505–527. Epub 2004 Dec 28. Bentin, T., Larsen, H. J., and Nielsen, P. E. (2003). Combined triplex/duplex invasion of doublestranded DNA by “tail-clamp” peptide nucleic acid. Biochemistry 42(47), 13987–13995. Bentin, T., and Nielsen, P. E. (1996). Enhanced peptide nucleic acid binding to supercoiled DNA: Possible implications for DNA “breathing” dynamics. Biochemistry 35(27), 8863–8869. Bentin, T., and Nielsen, P. E. (2003). Superior duplex DNA strand invasion by acridine conjugated peptide nucleic acids. J. Am. Chem. Soc. 125(21), 6378–6379. Boffa, L. C., Scarfi, S., Mariani, M. R., Damonte, G., Allfrey, V. G., Benatti, U., and Morris, P. L. (2000). Dihydrotestosterone as a selective cellular/nuclear localization vector for anti-gene peptide nucleic acid in prostatic carcinoma cells. Cancer Res. 60(8), 2258–2262. Bonham, M. A., Brown, S., Boyd, A. L., Brown, P. H., Bruckenstein, D. A., Hanvey, J. C., Thomson, S. A., Pipe, A., Hassman, F., and Bisi, J. E. (1995). An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers. Nucleic Acids Res. 23(7), 1197–1203. Borgatti, M., Lampronti, I., Romanelli, A., Pedone, C., Saviano, M., Bianchi, N., Mischiati, C., and Gambari, R. (2003a). Transcription factor decoy molecules based on a peptide nucleic acid (PNA)-DNA chimera mimicking Sp1 binding sites. J. Biol. Chem. 278(9), 7500–7509. Borgatti, M., Romanelli, A., Saviano, M., Pedone, C., Lampronti, I., Breda, L., Nastruzzi, C., Bianchi, N., Mischiati, C., and Gambari, R. (2003b). Resistance of decoy PNA-DNA chimeras to enzymatic degradation in cellular extracts and serum. Oncol. Res. 13(5), 279–287. Boules, M., Williams, K., Gollatz, E., Fauq, A., and Richelson, E. (2004). Down-regulation of amyloid precursor protein by peptide nucleic acid in vivo. J. Mol. Neurosci. 24(1), 123–128. Brande´n, L. J., Christensson, B., and Smith, C. I. (2001). In vivo nuclear delivery of oligonucleotides via hybridizing bifunctional peptides. Gene Ther. 8(1), 84–87. Brande´n, L. J., Mohamed, A. J., and Smith, C. I. (1999). A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA. Nat. Biotechnol. 17(8), 784–787. Brande´n, L. J., and Smith, C. I. (2002). Bioplex technology: Novel synthetic gene delivery system based on peptides anchored to nucleic acids. Methods Enzymol. 346, 106–124. Breipohl, G., Knolle, J., Langner, D., O’Malley, G., and Uhlmann, E. (1996). Synthesis of polyamide nucleic acids (PNAs) using a novel Fmoc/Mmt protecting-group combination. Bioorganic & Medicinal Chemistry Letters. 6(6), 665–670.

40

Lundin et al.

Bremner, K. H., Seymour, L. W., Logan, A., and Read, M. L. (2004). Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery. Bioconjug. Chem. 15 (1), 152–161. Brown, S. C., Thomson, S. A., Veal, J. M., and Davis, D. G. (1994). NMR solution structure of a peptide nucleic acid complexed with RNA. Science 265(5173), 777–780. Buchardt, O., Egholm, M., Nielsen, P. E., and Berg, R. H. (1992). Peptide nucleic acids. In “PCT Int. Appl., Wo 92/20702,” p. 192. Bukanov, N. O., Demidov, V. V., Nielsen, P. E., and Frank-Kamenetskii, M. D. (1998). PD-loop: A complex of duplex DNA with an oligonucleotide. Proc. Natl. Acad. Sci. USA 95(10), 5516–5520. Chaubey, B., Tripathi, S., Ganguly, S., Harris, D., Casale, R. A., and Pandey, V. N. (2005). A PNAtransportan conjugate targeted to the TAR region of the HIV-1 genome exhibits both antiviral and virucidal properties. Virology 331(2), 418–428. Clivio, P., Guillaume, D., Adeline, M.-T., Hamon, J., Riche, C., and Fourrey, J.-L. (1998). Synthesis and photochemical behavior of peptide nucleic acid dimers and analogs containing 4-Thiothymine: Unprecedented (5-4) photoadduct reversion. J. Am. Chem. Soc. 120(6), 1157–1166. Coull, J. M., Egholm, M., Hodge, R. P., Ismail, M., and Rajur, S. B. (1996). Improved synthons for the synthesis and deprotection of peptide nucleic acids under mild conditions. In “PCT Int. Appl. Wo 96/40685,” p. 138. Perseptive Biosystems Inc., USA. Cundliffe, E. (1987). On the nature of antibiotic binding sites in ribosomes. Biochimie 69(8), 863–869. Cutrona, G., Carpaneto, E. M., Ponzanelli, A., Ulivi, M., Millo, E., Scarfi, S., Roncella, S., Benatti, U., Boffa, L. C., and Ferrarini, M. (2003). Inhibition of the translocated c-myc in Burkitt’s lymphoma by a PNA complementary to the E mu enhancer. Cancer Res. 63(19), 6144–6148. Cutrona, G., Carpaneto, E. M., Ulivi, M., Roncella, S., Landt, O., Ferrarini, M., and Boffa, L. C. (2000). Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat. Biotechnol. 18(3), 300–303. Datta, B., Wang, M., and Armitage, B. A. (2004). PNA beacons and fluoroprobes. In “Peptoide Nucleic Acids, Protocols and Applications” (P. E. Nielsen, ed.), pp. 141–152. Horizon Bioscience, Wymondham. D’Costa, M., Kumar, V. A., and Ganesh, K. N. (1999). Aminoethylprolyl peptide nucleic acids (aepPNA): Chiral PNA analogues that form highly stable DNA: aepPNA2 triplexes. Org. Lett. 1(10), 1513–1516. de Koning, M. C., van der Marel, G. A., and Overhand, M. (2003). Synthetic developments towards PNA-peptide conjugates. Curr. Opin. Chem. Biol. 7(6), 734–740. De Mesmaeker, A., Waldner, A., Lebreton, J., Fritsch, V., and Wolf, R. M. (1994). Novel backbone replacements for oligonucleotides. ACS Symposium Series 580 (Carbohydrate Modifications in Antisense Research), pp. 24–39. American Chemical Society, Washington DC. Demidov, V., Frank-Kamenetskii, M. D., Egholm, M., Buchardt, O., and Nielsen, P. E. (1993). Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1. Nucleic Acids Res. 21(9), 2103–2107. Demidov, V. V., Potaman, V. N., Frank-Kamenetskii, M. D., Egholm, M., Buchard, O., Sonnichsen, S. H., and Nielsen, P. E. (1994). Stability of peptide nucleic acids in human serum and cellular extracts. Biochem. Pharmacol. 48(6), 1310–1313. Demidov, V. V., Protozanova, E., Izvolsky, K. I., Price, C., Nielsen, P. E., and Frank-Kamenetskii, M. D. (2002). Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids. Proc. Natl. Acad. Sci. USA 99(9), 5953–5958. Derossi, D., Chassaing, G., and Prochiantz, A. (1998). Trojan peptides: The penetratin system for intracellular delivery. Trends Cell Biol. 8(2), 84–87.

1. Biological and Biotechnological Aspects of PNA

41

Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994). The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269(14), 10444–10450. Dias, N., Senamaud-Beaufort, C., Forestier, EI. E., Auvin, C., Helene, C., and Ester SaisonBehmoaras, T. (2002). RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer. J. Mol. Biol. 320(3), 489–501. Doyle, D. F., Braasch, D. A., Simmons, C. G., Janowski, B. A., and Corey, D. R. (2001). Inhibition of gene expression inside cells by peptide nucleic acids: Effect of mRNA target sequence, mismatched bases, and PNA length. Biochemistry 40(1), 53–64. Dryselius, R., Aswasti, S. K., Rajarao, G. K., Nielsen, P. E., and Good, L. (2003). The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli. Oligonucleotides 13(6), 427–433. Dryselius, R., Nekhotiaeva, N., and Good, L. (2005). Antimicrobial synergy between mRNA- and protein-level inhibitors. J. Antimicrob. Chemother. 56(1), 97–103. Dueholm, K. L., Egholm, M., Behrens, C., Christensen, L., Hansen, H. F., Vulpius, T., Petersen, K. H., Berg, R. H., Nielsen, P. E., and Buchardt, O. (1994). Synthesis of peptide nucleic acid monomers containing the four natural nucleobases: Thymine, cytosine, adenine, and guanine and their oligomerization. J. Org. Chem. 59(19), 5767–5773. Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Norden, B., and Nielsen, P. E. (1993). PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365(6446), 566–568. Egholm, M., Christensen, L., Dueholm, K. L., Buchardt, O., Coull, J., and Nielsen, P. E. (1995). Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Res. 23(2), 217–222. Eriksson, M., and Nielsen, P. E. (1996). Solution structure of a peptide nucleic acid-DNA duplex. Nat. Struct. Biol. 3(5), 410–413. Eriksson, M., Nielsen, P. E., and Good, L. (2002). Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli. J. Biol. Chem. 277(9), 7144–7147. Fader, L. D., Boyd, M., and Tsantrizos, Y. S. (2001). Backbone modifications of aromatic peptide nucleic acid (APNA) monomers and their hybridization properties with DNA and RNA. J. Org. Chem. 66(10), 3372–3379. Fader, L. D., Myers, E. L., and Tsantrizos, Y. S. (2004). Synthesis of novel analogs of aromatic peptide nucleic acids (APNAs) with modified conformational and electrostatic properties. Tetrahedron 60(10), 2235–2246. Fader, L. D., and Tsantrizos, Y. S. (2002). Hybridization properties of aromatic peptide nucleic acids: A novel class of oligonucleotide analogues. Org. Lett. 4(1), 63–66. Farese, A., Patino, N., Condom, R., Dalleu, S., and Guedj, R. (1996). Liquid phase synthesis of a peptidic nucleic acid dimer. Tetrahedron Lett. 37(9), 1413–1416. Faruqi, A. F., Egholm, M., and Glazer, P. M. (1998). Peptide nucleic acid-targeted mutagenesis of a chromosomal gene in mouse cells. Proc. Natl. Acad. Sci. USA 95(4), 1398–1403. Filipovska, A., Eccles, M. R., Smith, R. A., and Murphy, M. P. (2004). Delivery of antisense peptide nucleic acids (PNAs) to the cytosol by disulphide conjugation to a lipophilic cation. FEBS Lett. 556(1–3), 180–186. Finn, P. J., Gibson, N. J., Fallon, R., Hamilton, A., and Brown, T. (1996). Synthesis and properties of DNA-PNA chimeric oligomers. Nucleic Acids Res. 24(17), 3357–3363. Fisher, L., Soomets, U., Cortes Toro, V., Chilton, L., Jiang, Y., Langel, U., and Iverfeldt, K. (2004). Cellular delivery of a double-stranded oligonucleotide NFkappaB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide. Gene Ther. 11(16), 1264–1272.

42

Lundin et al.

Folini, M., Berg, K., Millo, E., Villa, R., Prasmickaite, L., Daidone, M. G., Benatti, U., and Zaffaroni, N. (2003). Photochemical internalization of a peptide nucleic acid targeting the catalytic subunit of human telomerase. Cancer Res. 63(13), 3490–3494. Frank-Kamenetskii, M. D., and Demidov, V. V. (2004). PNA directed genome rare cutting: New developements. In “Peptoide Nucleic Acids, Protocols and Applications” (P. E. Nielsen, ed.), pp. 187–205. Horizon Bioscience, Wymondham. Fraser, G. L., Holmgren, J., Clarke, P. B., and Wahlestedt, C. (2000). Antisense inhibition of delta-opioid receptor gene function in vivo by peptide nucleic acids. Mol. Pharmacol. 57(4), 725–731. Gaborik, Z., and Hunyady, L. (2004). Intracellular trafficking of hormone receptors. Trends Endocrinol. Metab. 15(6), 286–293. Ganesh, K. N., and Nielsen, P. E. (2000). Peptide nucleic acids: Analogs and derivatives. Curr. Org. Chem. 4(9), 931–943. Gangamani, B. P., DeCosta, M., Kumar, V. A., and Ganesh, K. N. (1999a). Conformationally restrained chiral PNA conjugates: Synthesis and DNA complementation studies. Nucleosides Nucleotides. 18(6–7), 1409–1411. Gangamani, B. P., Kumar, V. A., and Ganesh, K. N. (1996). Synthesis of Na-(purinyl/pyrimidinyl acetyl)-4-aminoproline diastereomers with potential use in PNA synthesis. Tetrahedron 52(47), 15017–15030. Gangamani, B. P., Kumar, V. A., and Ganesh, K. N. (1997). Spermine conjugated peptide nucleic acids (spPNA): UV and fluorescence studies of PNA-DNA hybrids with improved stability. Biochem. Biophys. Res. Commun. 240(3), 778–782. Gangamani, B. P., Kumar, V. A., and Ganesh, K. N. (1999b). Chiral analogs of peptide nucleic acids: Synthesis of 4-aminoprolyl nucleic acids and DNA complementation studies using UV/CD spectroscopy. Tetrahedron 55(1), 177–192. Garner, P., Dey, S., and Huang, Y. (2000). a-Helical peptide nucleic acids (aPNAs): A new paradigm for DNA-binding molecules. J. Am. Chem. Soc. 122(10), 2405–2406. Garner, P., Dey, S., Huang, Y., and Zhang, X. (1999). Modular nucleic acid surrogates. Solid phase synthesis of a-helical peptide nucleic acids (aPNAs). Org. Lett. 1(3), 403–405. Garner, P., Huang, Y., and Dey, S. (2001). Enhancement of aPNA binding affinity and specificity through hydrophobic interactions. Chembiochem 2(3), 224–226. Geller, B. L., Deere, J., Tilley, L., and Iversen, P. L. (2005). Antisense phosphorodiamidate morpholino oligomer inhibits viability of Escherichia coli in pure culture and in mouse peritonitis. J. Antimicrob. Chemother. 55(6), 983–988. Giesen, U., Kleider, W., Berding, C., Geiger, A., Orum, H., and Nielsen, P. E. (1998). A formula for thermal stability (Tm) prediction of PNA/DNA duplexes. Nucleic Acids Res. 26(21), 5004–5006. Good, L., Awasthi, S. K., Dryselius, R., Larsson, O., and Nielsen, P. E. (2001). Bactericidal antisense effects of peptide-PNA conjugates. Nat. Biotechnol. 19(4), 360–364. Good, L., and Nielsen, P. E. (1998). Inhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNA. Proc. Natl. Acad. Sci. USA 95(5), 2073–2076. Good, L., Sandberg, R., Larsson, O., Nielsen, P. E., and Wahlestedt, C. (2000). Antisense PNA effects in Escherichia coli are limited by the outer-membrane LPS layer. Microbiology 146(Pt 10), 2665–2670. Govindaraju, T., Gonnade, R. G., Bhadbhade, M. M., Kumar, V. A., and Ganesh, K. N. (2003). (1S,2R/1R,2S)-Aminocyclohexyl glycyl thymine PNA: Synthesis, monomer crystal structures, and DNA/RNA hybridization studies. Org. Lett. 5(17), 3013–3016. Govindaraju, T., and Kumar, V. A. (2005). Backbone-extended pyrrolidine peptide nucleic acids (bepPNA): Design, synthesis and DNA/RNA binding studies. Chem. Commun. (Camb.) 4, 495–497.

1. Biological and Biotechnological Aspects of PNA

43

Govindaraju, T., Kumar, V. A., and Ganesh, K. N. (2004a). (1S,2R/1R,2S)-cis-cyclopentyl PNAs (cpPNAs) as constrained PNA analogs: Synthesis and evaluation of aeg-cpPNA chimera and stereopreferences in hybridization with DNA/RNA. J. Org. Chem. 69(17), 5725–5734. Govindaraju, T., Kumar, V. A., and Ganesh, K. N. (2004b). cis-Cyclopentyl PNA (cpPNA) as constrained chiral PNA analogues: Stereochemical dependence of DNA/RNA hybridization. Chem. Commun. (Camb.) 7, 860–861. Govindaraju, T., Kumar, V. A., and Ganesh, K. N. (2004c). Synthesis and evaluation of (1S,2R/ 1R,2S)-aminocyclohexylglycyl PNAs as conformationally preorganized PNA analogues for DNA/RNA recognition. J. Org. Chem. 69(6), 1858–1865. Govindaraju, T., Kumar, V. A., and Ganesh, K. N. (2005). (SR/RS)-cyclohexanyl PNAs: Conformationally preorganized PNA analogues with unprecedented preference for duplex formation with RNA. J. Am. Chem. Soc. 127(12), 4144–4145. Gray, G. D., Basu, S., and Wickstrom, E. (1997). Transformed and immortalized cellular uptake of oligodeoxynucleoside phosphorothioates, 30 -alkylamino oligodeoxynucleotides, 20 -O-methyl oligoribonucleotides, oligodeoxynucleoside methylphosphonates, and peptide nucleic acids. Biochem. Pharmacol. 53(10), 1465–1476. Guffanti, E., Corradini, R., Ottonello, S., and Dieci, G. (2004). Functional dissection of RNA polymerase III termination using a peptide nucleic acid as a transcriptional roadblock. J. Biol. Chem. 279(20), 20708–20716. Epub 2004 Feb 17. Hamilton, S. E., Pitts, A. E., Katipally, R. R., Jia, X., Rutter, J. P., Davies, B. A., Shay, J. W., Wright, W. E., and Corey, D. R. (1997). Identification of determinants for inhibitor binding within the RNA active site of human telomerase using PNA scanning. Biochemistry 36(39), 11873–11880. Hamilton, S. E., Simmons, C. G., Kathiriya, I. S., and Corey, D. R. (1999). Cellular delivery of peptide nucleic acids and inhibition of human telomerase. Chem. Biol. 6(6), 343–351. Hamzavi, R., Dolle, F., Tavitian, B., Dahl, O., and Nielsen, P. E. (2003a). Modulation of the pharmacokinetic properties of PNA: preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers. Bioconjug. Chem. 14(5), 941–954. Hamzavi, R., Dolle, F., Tavitian, B., Dahl, O., and Nielsen, P. E. (2003b). Modulation of the pharmacokinetic properties of PNA: Preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers. Bioconjug. Chem. 14(5), 941–954. Hanvey, J. C., Peffer, N. J., Bisi, J. E., Thomson, S. A., Cadilla, R., Josey, J. A., Ricca, D. J., Hassman, C. F., Bonham, M. A., Au, K. G., Carter, S. G., and Bruckenstein, D. A. (1992). Antisense and antigene properties of peptide nucleic acids. Science 258(5087), 1481–1485. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., et al. (2000). A Toll-like receptor recognizes bacterial DNA. Nature 408 (6813), 740–745. Hertoghs, K. M., Ellis, J. H., and Catchpole, I. R. (2003). Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA. Nucleic Acids Res. 31(20), 5817–5830. Hollenstein, M., and Leumann, C. J. (2003). Synthesis and incorporation into PNA of fluorinated olefinic PNA (F-OPA) monomers. Org. Lett. 5(11), 1987–1990. Hollenstein, M., and Leumann, C. J. (2005). Fluorinated olefinic peptide nucleic acid: Synthesis and pairing properties with complementary DNA. J. Org. Chem. 70(8), 3205–3217. Huang, Y., Dey, S., Zhang, X., Soennichsen, F., and Garner, P. (2004). The a-helical peptide nucleic acid concept: Merger of peptide secondary structure and codified nucleic acid recognition. J. Am. Chem. Soc. 126(14), 4626–4640. Ikeda, H., Nakamura, Y., and Saito, I. (2002). Synthesis and characterization of naphthalimidecontaining peptide nucleic acid. Tetrahedron Lett. 43(32), 5525–5528.

44

Lundin et al.

Ikeda, H., Yoshida, K., Ozeki, M., and Saito, I. (2001). Synthesis and characterization of flavintethered peptide nucleic acid. Tetrahedron Lett. 42(13), 2529–2531. Izvolsky, K. I., Demidov, V. V., Nielsen, P. E., and Frank-Kamenetskii, M. D. (2000). Sequencespecific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs. Biochemistry 39(35), 10908–10913. Jenuwein, T., and Allis, C. D. (2001). Translating the histone code. Science 293(5532), 1074–1080. Jiankuo, M., Xingbing, W., Baojun, H., Xiongwin, W., Zhuoya, L., Ping, X., Yong, X., Anting, L., Chunsong, H., Feili, G., and Jinguan, T. (2003). Peptide nucleic acid antisense prolongs skin allograft survival by means of blockade of CXCR3 expression directing T cells into graft. J. Immunol. 170(3), 1556–1565. Joliot, A., and Prochiantz, A. (2004). Transduction peptides: From technology to physiology. Nat. Cell Biol. 6(3), 189–196. Jordan, S., Schwemler, C., Kosch, W., Kretschmer, A., Schwenner, E., Stropp, U., and Mielke, B. (1997a). Synthesis of new building blocks for peptide nucleic acids containing monomers with variations in the backbone. Bioorg. Med. Chem. Lett. 7(6), 681–686. Jordan, S., Schwemler, C., Kosch, W., Kretschmer, A., Stropp, U., Schwenner, E., and Mielke, B. (1997b). New hetero-oligomeric peptide nucleic acids with improved binding properties to complementary DNA. Bioorg. Med. Chem. Lett. 7(6), 687–690. Kaihatsu, K., Huffman, K. E., and Corey, D. R. (2004). Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates. Biochemistry 43(45), 14340–14347. Kaihatsu, K., Shah, R. H., Zhao, X., and Corey, D. R. (2003). Extending recognition by peptide nucleic acids (PNAs): Binding to duplex DNA and inhibition of transcription by tail-clamp PNA-peptide conjugates. Biochemistry 42(47), 13996–14003. Kaplan, I. M., Wadia, J. S., and Dowdy, S. F. (2005). Cationic TAT peptide transduction domain enters cells by macropinocytosis. J. Control. Rel. 102(1), 247–253. Karras, J. G., Maier, M. A., Lu, T., Watt, A., and Manoharan, M. (2001). Peptide nucleic acids are potent modulators of endogenous pre-mRNA splicing of the murine interleukin-5 receptor-alpha chain. Biochemistry 40(26), 7853–7859. Karras, J. G., McKay, R. A., Dean, N. M., and Monia, B. P. (2000). Deletion of individual exons and induction of soluble murine interleukin-5 receptor-alpha chain expression through antisense oligonucleotide-mediated redirection of pre-mRNA splicing. Mol. Pharmacol. 58(2), 380–387. Kaushik, N., Basu, A., Palumbo, P., Myers, R. L., and Pandey, V. N. (2002). Anti-TAR polyamide nucleotide analog conjugated with a membrane-permeating peptide inhibits human immunodeficiency virus type 1 production. J. Virol. 76(8), 3881–3891. Kitamatsu, M., Shigeyasu, M., Okada, T., and Sisido, M. (2004). Oxy-peptide nucleic acid with a pyrrolidine ring that is configurationally optimized for hybridization with DNA. Chem. Commun. (Camb.) 10, 1208–1209. Knudsen, H., and Nielsen, P. E. (1996). Antisense properties of duplex- and triplex-forming PNAs. Nucleic Acids Res. 24(3), 494–500. Kohler, O., Jarikote, D. V., and Seitz, O. (2005). Forced intercalation probes (FIT Probes): Thiazole orange as a fluorescent base in peptide nucleic acids for homogeneous single-nucleotide-polymorphism detection. Chembiochem 6(1), 69–77. Komiyama, M., and Sumaoka, J. (1998). Progress towards synthetic enzymes for phosphoester hydrolysis. Curr. Opin. Chem. Biol. 2(6), 751–757. Komiyama, M., Ye, S., Liang, X., Yamamoto, Y., Tomita, T., Zhou, J. M., and Aburatani, H. (2003). PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection. J. Am. Chem. Soc. 125(13), 3758–3762.

1. Biological and Biotechnological Aspects of PNA

45

Koppelhus, U., Awasthi, S. K., Zachar, V., Holst, H. U., Ebbesen, P., and Nielsen, P. E. (2002). Celldependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates. Antisense Nucleic A 12(2), 51–63. Koppelhus, U., Zachar, V., Nielsen, P. E., Liu, X., Eugen-Olsen, J., and Ebbesen, P. (1997). Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA. Nucleic Acids Res. 25(11), 2167–2173. Kuan, J. Y., and Glazer, P. M. (2004). Targeted gene modification using triplex-forming oligonucleotides. Methods Mol. Biol. 262, 173–194. Kuhn, H., Demidov, V. V., Coull, J. M., Fiandaca, M. J., Gildea, B. D., and Frank-Kamenetskii, M. D. (2002). Hybridization of DNA and PNA molecular beacons to single-stranded and doublestranded DNA targets. J. Am. Chem. Soc. 124(6), 1097–1103. Kuhn, H., Demidov, V. V., Frank-Kamenetskii, M. D., and Nielsen, P. E. (1998). Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA. Nucleic Acids Res. 26(2), 582–587. Kuhn, H., Demidov, V. V., Gildea, B. D., Fiandaca, M. J., Coull, J. C., and Frank-Kamenetskii, M. D. (2001). PNA beacons for duplex DNA. Antisense Nucleic A 11(4), 265–270. Kuhn, H., Demidov, V. V., Nielsen, P. E., and Frank-Kamenetskii, M. D. (1999). An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA. J. Mol. Biol. 286(5), 1337–1345. Kuhn, H., Hu, Y., Frank-Kamenetskii, M. D., and Demidov, V. V. (2003). Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers. Biochemistry 42(17), 4985–4992. Kumagai, I., Takahashi, T., Hamasaki, K., Ueno, A., and Mihara, H. (2001). HIV Rev peptides conjugated with peptide nucleic acids and their efficient binding to RRE RNA. Bioorg. Med. Chem. Lett. 11(9), 1169–1172. Kumar, V. A., and Ganesh, K. N. (2005). Conformationally constrained PNA analogues: Structural evolution toward DNA/RNA binding selectivity. Acc. Chem. Res. 38(5), 404–412. Larsen, H. J., and Nielsen, P. E. (1996). Transcription-mediated binding of peptide nucleic acid (PNA) to double-stranded DNA: Sequence-specific suicide transcription. Nucleic Acids Res. 24(3), 458–463. Leijon, M., Graslund, A., Nielsen, P. E., Buchardt, O., Norden, B., Kristensen, S. M., and Eriksson, M. (1994). Structural characterization of PNA-DNA duplexes by NMR. Evidence for DNA in a B-like conformation. Biochemistry 33(33), 9820–9825. Liang, K. W., Hoffman, E. P., and Huang, L. (2000). Targeted delivery of plasmid DNA to myogenic cells via transferrin-conjugated peptide nucleic acid. Mol. Ther. 1(3), 236–243. Liu, B., Han, Y., Ferdous, A., Corey, D. R., and Kodadek, T. (2003). Transcription activation by a PNA-peptide chimera in a mammalian cell extract. Chem. Biol. 10(10), 909–916. Liu, Y., Braasch, D. A., Nulf, C. J., and Corey, D. R. (2004). Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids. Biochemistry 43(7), 1921–1927. Lohse, J., Dahl, O., and Nielsen, P. E. (1999). Double duplex invasion by peptide nucleic acid: A general principle for sequence-specific targeting of double-stranded DNA. Proc. Natl. Acad. Sci. USA 96, 11804–11808. Lundberg, M., and Johansson, M. (2001). Is VP22 nuclear homing an artifact? Nat. Biotechnol. 19(8), 713–714. Lundin, K. E., Ge, R., Svahn, M. G., Tornquist, E., Leijon, M., Branden, L. J., and Smith, C. I. (2004). Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras. Biomol. Eng. 21(2), 51–59. Lundin, K. E., Hasan, M., Moreno, P. M., To¨rnquist, E., Oprea, I., Svahn, M. G., Simonson, E. O., and Smith, C. I. E. (2005). Increased stability and specificity by combined peptide nucleic acid

46

Lundin et al.

(PNA) and locked nucleic acid (LNA) hybridization to super coiled plasmids for PNA-anchored “Bioplex” formation. Biomol. Eng. 22(5–6), 185–192. Marin, V. L., Roy, S., and Armitage, B. A. (2004). Recent advances in the development of peptide nucleic acid as a gene-targeted drug. Expert Opin. Biol. Ther. 4(3), 337–348. Mayhood, T., Kaushik, N., Pandey, P. K., Kashanchi, F., Deng, L., and Pandey, V. N. (2000). Inhibition of Tat-mediated transactivation of HIV-1 LTR transcription by polyamide nucleic acid targeted to TAR hairpin element. Biochemistry 39(38), 11532–11539. McMahon, B. M., Stewart, J., Fauq, A., Younkin, S., Younkin, L., and Richelson, E. (2003). Peptide nucleic acids targeted to the amyloid precursor protein. J. Mol. Neurosci. 20(3), 261–265. McMahon, B. M., Mays, D., Lipsky, J., Stewart, J. A., Fauq, A., and Richelson, E. (2002a). Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration. Antisense Nucleic A 12(2), 65–70. McMahon, B. M., Stewart, J. A., Bitner, M. D., Fauq, A., McCormick, D. J., and Richelson, E. (2002b). Peptide nucleic acids specifically cause antigene effects in vivo by systemic injection. Life Sci. 71(3), 325–337. Menchise, V., De Simone, G., Tedeschi, T., Corradini, R., Sforza, S., Marchelli, R., Capasso, D., Saviano, M., and Pedone, C. (2003). Insights into peptide nucleic acid (PNA) structural features: The crystal structure of a D-lysine-based chiral PNA-DNA duplex. Proc. Natl. Acad. Sci. USA 100(21), 12021–12026. Mischiati, C., Borgatti, M., Bianchi, N., Rutigliano, C., Tomassetti, M., Feriotto, G., and Gambari, R. (1999). Interaction of the human NF-kappaB p52 transcription factor with DNA-PNA hybrids mimicking the NF-kappaB binding sites of the human immunodeficiency virus type 1 promoter. J. Biol. Chem. 274(46), 33114–33122. Mokhir, A., Kraemer, R., and Wolf, H. (2004a). Zn2þ-dependent peptide nucleic acids probes. J. Am. Chem. Soc. 126(20), 6208–6209. Mokhir, A., Kramer, R., Voloshin, Y. Z., and Varzatskii, O. A. (2004b). Synthesis and DNA binding properties of dioxime-peptide nucleic acids. Bioorg. Med. Chem. Lett. 14(11), 2927–2930. Mokhir, A., Stiebing, R., and Kraemer, R. (2003a). Peptide nucleic acid-metal complex conjugates: Facile modulation of PNA-DNA duplex stability. Bioorg. Med. Chem. Lett. 13(8), 1399–1401. Mokhir, A., Zohm, B., Fuessl, A., and Kraemer, R. (2003b). Synthesis and DNA binding properties of terminally modified peptide nucleic acids. Bioorg. Med. Chem. Lett. 13(15), 2489–2492. Mollegaard, N. E., Buchardt, O., Egholm, M., and Nielsen, P. E. (1994). Peptide nucleic acid. DNA strand displacement loops as artificial transcription promoters. Proc. Natl. Acad. Sci. USA 91(9), 3892–3895. Morishita, R., Tomita, N., Kaneda, Y., and Ogihara, T. (2004). Molecular therapy to inhibit NFkappaB activation by transcription factor decoy oligonucleotides. Curr. Opin. Pharmacol. 4(2), 139–146. Moya, A., Holmes, E. C., and Gonzalez-Candelas, F. (2004). The population genetics and evolutionary epidemiology of RNA viruses. Nat. Rev. Microbiol. 2(4), 279–288. Munkonge, F. M., Dean, D. A., Hillery, E., Griesenbach, U., and Alton, E. W. (2003). Emerging significance of plasmid DNA nuclear import in gene therapy. Adv. Drug Deliv. Rev. 55(6), 749–760. Muratovska, A., Lightowlers, R. N., Taylor, R. W., Turnbull, D. M., Smith, R. A., Wilce, J. A., Martin, S. W., and Murphy, M. P. (2001). Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: Implications for mitochondrial DNA replication, expression and disease. Nucleic Acids Res. 29(9), 1852–1863. Myers, M. C., Witschi, M. A., Larionova, N. V., Franck, J. M., Haynes, R. D., Hara, T., Grakowski, A., and Appella, D. H. (2003). A cyclopentane conformational restraint for a peptide nucleic acid: Design, asymmetric synthesis, and improved binding affinity to DNA and RNA. Org. Lett. 5(15), 2695–2698.

1. Biological and Biotechnological Aspects of PNA

47

Nekhotiaeva, N., Awasthi, S. K., Nielsen, P. E., and Good, L. (2004). Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids. Mol. Ther. 10(4), 652–659. Nielsen, K. V., Muller, S., Poulsen, T. S., Gabs, S., and Schonau, A. (2004). Combined use of PNA and DNA for fluorescence in situ hybridization (FISH). In “Peptoide Nucleic Acids, Protocols and Applications” (P. E. Nielsen, ed.), pp. 227–260. Horizon Bioscience, Wymondham. Nielsen, P. (2003). The many faces of PNA. Lett. Pept. Sci. 10, 135–137. Nielsen, P. E. (2005). Gene targeting using peptide nucleic acid. Methods Mol. Biol. 288, 343–358. Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1991). Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497–1500. Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1993a). Peptide nucleic acids (PNAs): Potential antisense and anti-gene agents. Anticancer Drug Des. 8(1), 53–63. Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1993b). Sequence specific inhibition of DNA restriction enzyme cleavage by PNA. Nucleic Acids Res. 21(2), 197–200. Nielsen, P. E., Egholm, M., and Buchardt, O. (1994). Sequence-specific transcription arrest by peptide nucleic acid bound to the DNA template strand. Gene 149(1), 139–145. Nielsen, P. E., Frederiksen, K., and Behrens, C. (2005). Extended target sequence specificity of PNA-minor-groove binder conjugates. Chembiochem 6(1), 66–68. Nikaido, H. (2003). Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67(4), 593–656. Nina, M., Fonne-Pfister, R., Beaudegnies, R., Chekatt, H., Jung, P. M. J., Murphy-Kessabi, F., De Mesmaeker, A., and Wendeborn, S. (2005). Recognition of RNA by amide modified backbone nucleic acids: Molecular dynamics simulations of DNA-RNA hybrids in aqueous solution. J. Am. Chem. Soc. 127(16), 6027–6038. Noble, S. A., Binham, M. A., Bisi, J. E., Bruckenstein, D. A., Brown, P. H., Brown, S. C., Cadilla, R., Gaul, M. D., and Hanvey, J. C. (1995). Impact of biophysical parameters on the biological assessment of peptide nucleic acids, antisense inhibitors of gene expression. Drug Devlop. Res. 34, 184–195. Nulf, C. J., and Corey, D. (2004). Intracellular inhibition of hepatitis C virus (HCV) internal ribosomal entry site (IRES)-dependent translation by peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). Nucleic Acids Res. 32(13), 3792–3798. Ortiz, E., Estrada, G., and Lizardi, P. M. (1998). PNA molecular beacons for rapid detection of PCR amplicons. Mol. Cell. Probes 12(4), 219–226. Ørum, H. (2004). PCR clamping. In “Peptide Nucleic Acids, Protocols and Applications” (P. E. Nielsen, ed.), pp. 175–185. Horizon Bioscience, Wymondham. Pande, V., and Ramos, M. J. (2005). NF-kappaB in human disease: Current inhibitors and prospects for de novo structure based design of inhibitors. Curr. Med. Chem. 12(3), 357–374. Pardridge, W. M., Boado, R. J., and Kang, Y. S. (1995). Vector-mediated delivery of a polyamide (“peptide”) nucleic acid analogue through the blood-brain barrier in vivo. Proc. Natl. Acad. Sci. USA 92(12), 5592–5596. Paulasova, P., Andreo, B., Diblik, J., Macek, M., and Pellestor, F. (2004). The peptide nucleic acids as probes for chromosomal analysis: Application to human oocytes, polar bodies and preimplantation embryos. Mol. Hum. Reprod. 10(6), 467–472. Pellestor, F., Anahory, T., and Hamamah, S. (2005). The chromosomal analysis of human oocytes. An overview of established procedures. Hum. Reprod. Update 11(1), 15–32. Penolazzi, L., Borgatti, M., Lambertini, E., Mischiati, C., Finotti, A., Romanelli, A., Saviano, M., Pedone, C., Piva, R., and Gambari, R. (2004). Peptide nucleic acid-DNA decoy chimeras targeting NF-kappaB transcription factors: Induction of apoptosis in human primary osteoclasts. Int. J. Mol. Med. 14(2), 145–152.

48

Lundin et al.

Pesce, C. D., Bolacchi, F., Bongiovanni, B., Cisotta, F., Capozzi, M., Diviacco, S., Quadrifoglio, F., Mango, R., Novelli, G., and Mossa, G. (2005). Anti-gene peptide nucleic acid targeted to proviral HIV-1 DNA inhibits in vitro HIV-1 replication. Antiviral Res. 66(1), 13–22. Petersen, K., Vogel, U., Rockenbauer, E., Nielsen, K. V., Kolvraa, S., Bolund, L., and Nexo, B. (2004a). Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms. Mol. Cell. Probes 18(2), 117–122. Petersen, L., de Koning, M. C., van Kuik-Romeijn, P., Weterings, J., Pol, C. J., Platenburg, G., Overhand, M., van der Marel, G. A., and van Boom, J. H. (2004b). Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases. Bioconjug. Chem. 15(3), 576–582. Petersson, B., Nielsen, B. B., Rasmussen, H., Larsen, I. K., Gajhede, M., Nielsen, P. E., and Kastrup, J. S. (2005). Crystal structure of a partly self-complementary peptide nucleic acid (PNA) oligomer showing a duplex-triplex network. J. Am. Chem. Soc. 127(5), 1424–1430. Peyman, A., Uhlmann, E., Wagner, K., Augustin, S., Breipohl, G., Will, D. W., Schaefer, A., and Wallmeier, H. (1996). Phosphonic ester nucleic acids (PHONAs): Oligodeoxyribonucleotide analog with an achiral phosphonic acid ester backbone. Angew. Chem. Int. Ed. Engl. 35(22), 2636–2638. Pokorski, J. K., Myers, M. C., and Appella, D. H. (2005). Cyclopropane PNA: Observable triplex melting in a PNA constrained with a 3-membered ring. Tetrahedron Lett. 46(6), 915–917. Pokorski, J. K., Witschi, M. A., Purnell, B. L., and Appella, D. H. (2004). (S,S)-trans-cyclopentaneconstrained peptide nucleic acids. A general backbone modification that improves binding affinity and sequence specificity. J. Am. Chem. Soc. 126(46), 15067–15073. Pooga, M., Soomets, U., Hallbrink, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J. X., Xu, X. J., and Wiesenfeld-Hallin, Z. (1998). Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16(9), 857–861. Popescu, D.-L., Parolin, T. J., and Achim, C. (2003). Metal incorporation in modified PNA duplexes. J. Am. Chem. Soc. 125(21), 6354–6355. Rasmussen, H., Kastrup, J. S., Nielsen, J. N., Nielsen, J. M., and Nielsen, P. E. (1997). Crystal structure of a peptide nucleic acid (PNA) duplex at 1.7 A resolution. Nat. Struct. Biol. 4(2), 98–101. Rebufat, A. G., Bernasconi, A. G., and Frey, B. M. (2004). Vector tagging via PNA. In “Peptoide Nucleic Acids, Protocols and Applications” (P. E. Nielsen, ed.), pp. 153–174. Horizon Bioscience, Wymondham. Rebuffat, A. G., Nawrocki, A. R., Nielsen, P. E., Bernasconi, A. G., Bernal-Mendez, E., Frey, B. M., and Frey, F. J. (2002). Gene delivery by a steroid-peptide nucleic acid conjugate. FASEB J. 16(11), 1426–1428. Rembach, A., Turner, B. J., Bruce, S., Cheah, I. K., Scott, R. L., Lopes, E. C., Zagami, C. J., Beart, P. M., Cheung, N. S., and Langford, S. J. (2004). Antisense peptide nucleic acid targeting GluR3 delays disease onset and progression in the SOD1 G93A mouse model of familial ALS. J. Neurosci. Res. 77(4), 573–582. Rezaei, K., Xu, I. S., Wu, W. P., Shi, T. J., Soomets, U., Land, T., Xu, X. J., Wiesenfeld-Hallin, Z., Hokfelt, T., Bartfai, T., and Langel, V. (2001). Intrathecal administration of PNA targeting galanin receptor reduces galanin-mediated inhibitory effect in the rat spinal cord. Neuroreport 12 (2), 317–320. Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. (2003). Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278(1), 585–590. Riguet, E., Tripathi, S., Chaubey, B., Desire, J., Pandey, V. N., and Decout, J.-L. (2004). A peptide nucleic acid-Neamine conjugate that targets and cleaves HIV-1 TAR RNA inhibits viral replication. J. Med. Chem. 47(20), 4806–4809.

1. Biological and Biotechnological Aspects of PNA

49

Robaczewska, M., Narayan, R., Seigneres, B., Schorr, O., Thermet, A., Podhajska, A. J., Trepo, C., Zoulim, F., Nielsen, P. E., and Cova, L. (2005). Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA). J. Hepatol. 42(2), 180–187. Rogers, F. A., Vasquez, K. M., Egholm, M., and Glazer, P. M. (2002). Site-directed recombination via bifunctional PNA-DNA conjugates. Proc. Natl. Acad. Sci. USA 99(26), 16695–16700. Ross, G. F., Smith, P. M., McGregor, A., Turnbull, D. M., and Lightowlers, R. N. (2003). Synthesis of trifunctional PNA-benzophenone derivatives for mitochondrial targeting, selective DNA binding, and photo-cross-linking. Bioconjug. Chem. 14(5), 962–966. Rozners, E., Katkevica, D., Bizdena, E., and Stroemberg, R. (2003). Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions. J. Am. Chem. Soc. 125 (40), 12125–12136. Saetrom, P. (2004). Predicting the efficacy of short oligonucleotides in antisense and RNAi experiments with boosted genetic programming. Bioinformatics 20(17), 3055–3063. Salvi, J. P., Walchshofer, N., and Paris, J. (1994). Formation of bis(Fmoc-aminoethyl)-N-glycine derivatives by reductive amination of Fmoc amino aldehydes with NaBH3CN. Tetrahedron Lett. 35(8), 1181–1184. Saxena, S. K., and Ackerman, E. J. (1990). Microinjected oligonucleotides complementary to the alphasarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes. J. Biol. Chem. 265(6), 3263–3269. Sazani, P., Gemignani, F., Kang, S. H., Maier, M. A., Manoharan, M., Persmark, M., Bortner, D., and Kole, R. (2002). Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat. Biotechnol. 20(12), 1228–1233. Sazani, P., Kang, S. H., Maier, M. A., Wei, C., Dillman, J., Summerton, J., Manoharan, M., and Kole, R. (2001). Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res. 29(19), 3965–3974. Scherer, L. J., and Rossi, J. J. (2003). Approaches for the sequence-specific knockdown of mRNA. Nat. Biotechnol. 21(12), 1457–1465. Sei, S., Yang, Q. E., O’Neill, D., Yoshimura, K., Nagashima, K., and Mitsuya, H. (2000). Identification of a key target sequence to block human immunodeficiency virus type 1 replication within the gag-pol transframe domain. J. Virol. 74(10), 4621–4633. Seitz, O. (2000). Solid-phase synthesis of doubly labeled peptide nucleic acids as probes for the realtime detection of hybridization. Angew. Chem. Int. Ed. Engl. 39(18), 3249–3252. Sforza, S., Corradini, R., Ghirardi, S., Dossena, A., and Marchelli, R. (2000). DNA binding of a Dlysine-based chiral PNA: Direction control and mismatch recognition. Eur. J. Org. Chem. 16, 2905–2913. Sforza, S., Haaima, G., Marchelli, R., and Nielsen, P. E. (1999). Chiral peptide nucleic acids (PNAs). Helix handedness and DNA recognition. Eur. J. Org. Chem. 1, 197–204. Sharp, P. A. (2005). The discovery of split genes and RNA splicing. Trends Biochem. Sci. 30(6), 279–281. Shiraishi, T., and Nielsen, P. E. (2004). Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine-PNA (peptide nucleic acid) conjugates. Nucleic Acids Res. 32(16), 4893–4902. Sivori, S., Falco, M., Della Chiesa, M., Carlomagno, S., Vitale, M., Moretta, L., and Moretta, A. (2004). CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: Induction of cytokine release and cytotoxicity against tumors and dendritic cells. Proc. Natl. Acad. Sci. USA 101(27), 10116–10121. Slaitas, A., and Yeheskiely, E. (2001). Synthesis and hybridization of novel chiral pyrrolidine based PNA analogs. Nucleosides Nucleotides Nucleic Acids 20(4–7), 1377–1379. Slaitas, A., and Yeheskiely, E. (2002). A novel N-(pyrrolidinyl-2-methyl)glycine-based PNA with a strong preference for RNA over DNA. Eur. J. Org. Chem. 14, 2391–2399.

50

Lundin et al.

Stender, H. (2003). PNA FISH: An intelligent stain for rapid diagnosis of infectious diseases. Expert Rev. Mol. Diagn. 3(5), 649–655. Svahn, M. G., Lundin, K. E., Ge, R., Tornquist, E., Simonson, E. O., Oscarsson, S., Leijon, M., Branden, L. J., and Smith, C. I. (2004a). Adding functional entities to plasmids. J. Gene Med. 6 (Suppl 1), S36–S44. Svahn, M. G., Salih, G., Simonson, E. O., Smith, C. I., and Branden, L. J. (2004b). Protease-induced release of functional peptides from bioplexes. J. Control. Rel. 98(1), 169–177. Svanvik, N., Nygren, J., Westman, G., and Kubista, M. (2001). Free-probe fluorescence of light-up probes. J. Am. Chem. Soc. 123(5), 803–809. Svanvik, N., Westman, G., Wang, D., and Kubista, M. (2000). Light-up probes: Thiazole orangeconjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution. Anal. Biochem. 281(1), 26–35. Tan, X. X., Actor, J. K., and Chen, Y. (2005). Peptide nucleic acid antisense oligomer as a therapeutic strategy against bacterial infection: Proof of principle using mouse intraperitoneal infection. Antimicrob. Agents Chemother. 49(8), 3203–3207. Thierry, A. R., Vives, E., Richard, J. P., Prevot, P., Martinand-Mari, C., Robbins, I., and Lebleu, B. (2003). Cellular uptake and intracellular fate of antisense oligonucleotides. Curr. Opin. Mol. Ther. 5(2), 133–138. Thomson, S. A., Josey, J. A., Cadilla, R., Gaul, M. D., Hassman, C. F., Luzzio, M. J., Pipe, A. J., Reed, K. L., and Ricca, D. J. (1995). Fmoc mediated synthesis of peptide nucleic acids. Tetrahedron 51(22), 6179–6194. Tomac, S., Sarkar, M., Ratilainen, T., Wittung, P., Nielsen, P., Norde´n, B., and Gra¨slund, A. (1996). Ionic effects on the stability and conformation of peptide nucleic acid (PNA) complexes. J. Am. Chem. Soc. 118, 5544–5552. Tripathi, S., Chaubey, B., Ganguly, S., Harris, D., Casale, R. A., and Pandey, V. N. (2005). AntiHIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides. Nucleic Acids Res. 33(13), 4345–4356. Tsantrizos, Y. S., Lunetta, J. F., Boyd, M., Fader, L. D., and Wilson, M.-C. (1997). Stereoselective synthesis of a thymine derivative of (S)-2-hydroxy-4-(2-aminophenyl)butanoic acid. A novel building block for the synthesis of aromatic peptidic nucleic acid oligomers. J. Org. Chem. 62(16), 5451–5457. Tyagi, S., and Kramer, F. R. (1996). Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14(3), 303–308. Tyler, B. M., Jansen, K., McCormick, D. J., Douglas, C. L., Boules, M., Stewart, J. A., Zhao, L., Lacy, B., Cusack, B., Fauq, A., and Richelson, E. (1999). Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood-brain barrier and specifically reduce gene expression. Proc. Natl. Acad. Sci. USA 96(12), 7053–7058. Tyler, B. M., McCormick, D. J., Hoshall, C. V., Douglas, C. L., Jansen, K., Lacy, B. W., Cusack, B., and Richelson, E. (1998). Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo. FEBS Lett. 421(3), 280–284. Uhlmann, E., Will, D. W., Breipohl, G., Langner, D., and Ryte, A. (1996). Synthesis and properties of PNA/DNA chimeras. Angew. Chem. Int. Ed. Engl. 35(22), 2632–2635. van der Laan, A. C., Meeuwenoord, N. J., Kuyl-Yeheskiely, E., Oosting, R. S., Brands, R., and van Boom, J. H. (1995). Solid support synthesis of a PNA-DNA hybrid. Recueil des Travaux Chimiques des Pays-Bas. 114(6), 295–297. van der Laan, A. C., Stromberg, R., van Boom, J. H., Kuyl-Yeheskiely, E., Efimov, V. A., and Chakhmakhcheva, O. G. (1996). An approach towards the synthesis of oligomers containing a N-2-hydroxyethyl-aminomethylphosphonate backbone: A novel PNA analog. Tetrahedron Lett. 37(43), 7857–7860.

1. Biological and Biotechnological Aspects of PNA

51

Verheijen, J. C., Deiman, B. A. L. M., Yeheskiely, E., Van der Marel, G. A., and Van Boom, J. H. (2000). Efficient hydrolysis of RNA by a PNA-diethylenetriamine adduct. Angew. Chem. Int. Ed. Engl. 39(2), 369–372. Vernille, J. P., Kovell, L. C., and Schneider, J. W. (2004). Peptide nucleic acid (PNA) amphiphiles: Synthesis, self-assembly, and duplex stability. Bioconjug. Chem. 15(6), 1314–1321. Veselkov, A. G., Demidov, V. V., Frank-Kamenetskii, M. D., and Nielsen, P. E. (1996). PNA as a rare genome-cutter. Nature 379(6562), 214. Vilaivan, T., and Lowe, G. (2002). A novel pyrrolidinyl PNA showing high sequence specificity and preferential binding to DNA over RNA. J. Am. Chem. Soc. 124(32), 9326–9327. Vives, E., Brodin, P., and Lebleu, B. (1997). A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272(25), 16010–16017. Wang, G., Jing, K., Balczon, R., and Xu, X. (2001). Defining the peptide nucleic acids (PNA) length requirement for PNA binding-induced transcription and gene expression. J. Mol. Biol. 313(5), 933–940. Wang, G., Seidman, M. M., and Glazer, P. M. (1996). Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science 271(5250), 802–805. Wang, G., Xu, X., Pace, B., Dean, D. A., Glazer, P. M., Chan, P., Goodman, S. R., and Shokolenko, I. (1999). Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression. Nucleic Acids Res. 27(13), 2806–2813. Wilhelmsson, L. M., Norden, B., Mukherjee, K., Dulay, M. T., and Zare, R. N. (2002). Genetic screening using the colour change of a PNA-DNA hybrid-binding cyanine dye. Nucleic Acids Res. 30(2), E3. Wittekindt, C., Bottger, M., Holtje, H. D., Schafer-Korting, M., Cartier, R., and Haberland, A. (2004). Integrin specificity of the cyclic Arg-Gly-Asp motif and its role in integrin-targeted gene transfer. Biotechnol. Appl. Biochem. 40(Pt 3), 281–290. Wittung, P., Kajanus, J., Edwards, K., Haaima, G., Nielsen, P. E., Norden, B., and Malmstrom, B. G. (1995). Phospholipid membrane permeability of peptide nucleic acid. FEBS Lett. 375(3), 27–29. Wolffs, P., Knutsson, R., Sjoback, R., and Radstrom, P. (2001). PNA-based light-up probes for realtime detection of sequence-specific PCR products. Biotechniques 31(4), 766, 769–771. Wu, J., Nantz, M. H., and Zern, M. A. (2002). Targeting hepatocytes for drug and gene delivery: Emerging novel approaches and applications. Front. Biosci. 7, d717–d725. Xu, X. S., Glazer, P. M., and Wang, G. (2000). Activation of human gamma-globin gene expression via triplex-forming oligonucleotide (TFO)-directed mutations in the gamma-globin gene 50 flanking region. Gene 242(1–2), 219–228. Yamamoto, Y., Uehara, A., Tomita, T., and Komiyama, M. (2004). Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA. Nucleic Acids Res. 32(19), e153. Zelder, F. H., Mokhir, A. A., and Kramer, R. (2003). Sequence selective hydrolysis of linear DNA using conjugates of Zr(IV) complexes and peptide nucleic acids. Inorg. Chem. 42(26), 8618–8620. Zelphati, O., Liang, X., Nguyen, C., Barlow, S., Sheng, S., Shao, Z., and Felgner, P. L. (2000). PNAdependent gene chemistry: Stable coupling of peptides and oligonucleotides to plasmid DNA. Biotechniques 28(2), 304–310, 312–314, 316. Zhang, X., Simmons, C. G., and Corey, D. R. (2001). Liver cell specific targeting of peptide nucleic acid oligomers. Bioorg. Med. Chem. Lett. 11(10), 1269–1272. Zhou, P., Wang, M., Du, L., Fisher, G. W., Waggoner, A., and Ly, D. H. (2003). Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA). J. Am. Chem. Soc. 125(23), 6878–6879.

This page intentionally left blank

2

Changing Images of the Gene George P. Re´dei,* Csaba Koncz,† and Jane D. Phillips‡ *University of Missouri, Columbia, Missouri 65203 † Max-Planck-Institut, D-59829 Ko¨ln, Germany ‡ Life Sciences Center, University of Missouri, Columbia, Missouri 65211

I. II. III. IV.

V. VI.

VII. VIII. IX. X. XI.

XII. XIII. XIV.

Introduction The Hypothetical Gene Physical Concepts of the Gene Chemical Nature of the Gene A. Uncertain substance of the hereditary material B. The DNA-double helix C. RNA genes The Genetic Code Structure of the Gene A. Step allelomorphism and pseudoalleles B. Transposable elements C. Nonsexual transfer of genes Molecular Units of the Gene Higher Orders of Genes Gene Number and Gene Size Genes in the Cytoplasm Epigenetics A. Paramutation B. Imprinting C. Mechanisms of epigenetic modifications Prions Conclusions and Outlook Epilog Acknowledgments References

Advances in Genetics, Vol. 56 Copyright 2006, Elsevier Inc. All rights reserved.

0065-2660/06 $35.00 DOI: 10.1016/S0065-2660(06)56002-X

54

Re´dei et al.

ABSTRACT During the twentieth century the gene emerged as the major driving force of biology. Initially, even the nature and behavior of gene vehicles, the chromosomes, were subjected to doubts. The basic or standard gene concept, as a unit of function, mutation, and recombination, had to be revised. Half a century was required for reaching a general consensus about the chemical nature of the genetic material, DNA and RNA. The relationship between single genes and individual proteins was a great milestone at the middle of the twentieth century, but within two decades it was realized that the relationship was more complex. Understanding of genetic coding, transcription, and translation during the 1960s laid a firm foundation to the “nucleic doctrine,” harking back to the dicta of Lederberg (1959) and meaning that single nucleic acid genes alone were responsible for each separate function within the cell. However, important aspects of gene expression are recognized now as a function of the genome and many genes collaborate in circuits. It has come to light that genes may be mobile, exist in plasmids and cytoplasmic organelles, and can be imported by nonsexual means from other organisms or as synthetic products. Epigenetics has reborn as a new field of developmental genetics. The unorthodox prion proteins can even simulate some gene properties. Genetics was to an extent reincarnated as of the twenty-first century by assimilating the tools of cybernetics and of many formerly distant areas of science. This overview highlights some of the historical milestones that contributed to the development of our image of the gene, extending elements of issues laid down by Re´dei (2003). ß 2006, Elsevier Inc.

I. INTRODUCTION Although the term gene was conceived in 1909, for decades it was an abstraction without any physical meaning. The great pioneers of genetics, Johannsen (1909) and Bateson (1926), were reluctant to accept any material basis for it. Bateson died in 1922 without recognizing the general validity of the chromosome theory for plants, although he admitted its possible relevance for animals. On the contrary, Thomas Hunt Morgan, the founder and major figure of classic genetics, felt as early as the 1920s that cytogenetics had already exhausted its potentials (Borsook, 1956). Although Friedrich Miescher, a contemporary of Mendel, described nucleic acids in 1871, there remained an almost century-long disconnect between his findings and those of Mendel. It is interesting to note that the nature of the mechanism of fertilization remained an enigma even to Miescher. “So werden wir von allen seiten geno¨thigt, es mit Bestimmheit auszusprechen: Es giebt keine spezifischen Befruchtungsstoffe. Die chemischen Thatsachen haben

2. Changing Images of the Gene

55

secunda¨re Bedeutung; sie sind einem ho¨heren Gesichtspunkt untergeordnet.” (Miescher, 1874). [There is no specific fertilization-substance. The chemical realities have secondary meaning; they are subject to a higher order of strategy.] It is remarkable that the field of genetics was successful in building a vast amount of knowledge on the basis of limited facts and sharp logic. Mendel’s rules were based on probability and (indirectly or implicitly) on meiosis, although he did not know about genes and chromosomes (Strasburger, 1875) in the modern sense. Reductional division (during meiosis) was discovered only in 1883—by Van Beneden in Ascaris—after the conclusion of Mendel’s experiments in 1865. The discovery of bacterial transformation in 1928–1944 and construction of a model of DNA in 1953 would have been expected to change the perception of the gene immediately. However, even these facts were followed by certain apperception lags. A quantum leap occurred during the second half of the twentieth century, when from the early “molecular chaos” (Troland, 1917) a new beacon of science emerged. At the height of the golden age, after the basic elements of nucleic acid structure and function were elucidated, some believed again that there was not too much more to be learned. Some geneticists, particularly Gunther Stent (1969), predicted “the end to progress” since “the hope that paradoxes might still turn up in the study of heredity had to be abandoned long ago” (p. 66 within Stent’s book The coming of the Golden Age). Others, such as Sydney Brenner (1993), believed that “... genetics will disappear as a separate science because, in the 21st century, everything in biology will be gene based.” In fact, the latter view was a realistic forecast based on the evolution of the gene concept.

II. THE HYPOTHETICAL GENE One of the first important steps in evolution of the gene concept is associated with the groundbreaking discoveries of L. J. Stadler—and others—in the analysis of induced mutations. In 1942, Stadler demonstrated that the mutagenic effectiveness of UV-irradiation coincided with the absorption spectrum of nucleic acids (Stadler and Uber, 1942). Among the several kinds of gene studies performed by Stadler were those that indicated the “compound” nature of certain genes (Re´dei, 1971). Yet, Stadler (1954) expressed a rather pessimistic view, writing in 1954 that: “The difficulties in the study of the genic substance are obvious. It cannot be isolated for chemical analysis or pure culture. The possibility of direct analysis of specific segments or individual genes is, of course, even more remote. The properties of the genes may be inferred only from the results of their action.” Interestingly, H. J. Muller (1950), the second geneticist who received the Nobel Prize for the discovery of induction of mutation by

56

Re´dei et al.

X-rays, also shared this general opinion. It is therefore not surprising that in his last publication—The Gene, Stadler (1954)—did not refer to Watson and Crick (1953a), whose now famous paper appeared about a year before Stadler’s death. At mid-twentieth century, many leading geneticists were preoccupied with pseudoallelism (Lewis, 1945) and with the one gene–one enzyme concept (Beadle, 1945). The latter theory provided a means to exploration of gene function and expression, whereas pseudoallelism questioned the validity of the idea that the gene is an indivisible bead on a given chromosomal “string” (Oliver, 1940). During the predouble-helix period, the latter theory was supported by E. Schro¨dinger, who suggested that the chromosome is an aperiodic crystal. This physical idea was widely hailed, but geneticists did not have the technology for the needed experimental studies. According to Schro¨dinger (1967, p. 65): “The aperiodic crystal is like a masterpiece of embroidery, say a Raphael tapestry, which shows no dull repetition, but an elaborate, coherent, meaningful design traced by the great master”. . . . “For illustration, think of the Morse code. The two different signs of dot and dash in well-ordered groups of not more than four allow of thirty different specifications.” The periodic crystal is like “ordinary wallpaper in which the same pattern is repeated again and again in regular periodicity.” Schro¨dinger (1967, p. 31) accepted Darlington’s guesstimate of 2000 bands in the salivary chromosomes of Drosophila and considered this number the same as the number of genes. Dividing 2000 by the length of the ˚ .” A chromosomes, he suggested that “a gene [is] equal to cube of edge 300 A “gene contains certainly not more than about a million or a few million atoms.” Based on this, he concluded that the gene “is probably a large protein molecule, in which every atom, every radical, every heterocyclic ring plays an individual role, more or less different from that played by any of the other similar atoms, radicals or rings. This, at any rate, is the opinion of leading geneticists, such as Haldane and Darlington.” Schro¨dinger admitted that he was not “a master” of genetics: “a poor theoretical physicist could not be expected to produce anything like a competent survey of the experimental facts.” Nevertheless, after receiving the Nobel Prize in physics “for the discovery of new productive forms of atomic theory” in 1933, Schro¨dinger’s book (1967; published first in 1943) on physical aspects of living cells became one of the most stimulating and popular publications. In fact, the work of Timofe´eff-Ressovfsky et al. (1935), who explored the mutational hit theory, supported to elements of Schro¨dinger’s ideas. Stadler (1954), who also cited this theory, felt that their conjecture regarding the size and nature of genes has no basis in reality. Obviously, Stadler did not argue against the molecular reality of the gene. Rather, he merely cautioned against running ahead of solid experimental evidence. He warned that equating the facts of mutant phenotypes with interpretation of alterations within genes “we risk confusing what we know with what we only think we know.” Perhaps, Stadler’s position was

2. Changing Images of the Gene

57

also influenced by Timofe´eff-Ressovsky’s conclusions (1930) on forward and reverse mutations of Drosophila genes by X-ray induction that were not generally reproducible by others. Stadler had a firm conviction that X-rays have only destructive effects on genes (Stadler and Roman, 1948). This view was in sharp contrast to the experience with Neurospora (Giles, 1951) and of some Drosophila workers, including the laboratory of H. J. Muller (Hanson, 1928). [It is important to note here that Nicolay Timofe´eff-Ressovsky cannot be judged on the negative aspects of these experiments, because his striving (with Max Delbru¨ck and K. G. Zimmer) to define the corpuscular gene was a major milestone in the quest for the physical nature of the gene. The bison, as some of his Russian friends named him because of his robust stature, was a great biologist. Even after his abduction by the Red Army following the fall of Berlin-Dahlem at the end of the Second World War, he entertained his fellow inmates with his brilliant lectures about genes and the nature of the atom as the intellectual “President of Cell 75” in the Butryki prison (Solzhenitsyn, 1973).] Unlike many other geneticists, Stadler (1954) was in favor of distinguishing the operational concept of the gene that can be defined by its visible function and by the structural concept that he could only study by recombination. Stadler’s concept was supported by his extensive studies of the R locus of maize (Stadler, 1946). Using genetically and cytologically marked chromosomes, Stadler recognized that mutational changes were accompanied by recombination. By studying recombination at the R locus using (P) alleles, affecting embryo color, and (S) alleles, controlling aleuron (seed) color, Stadler found evidence for occurrence of unequal crossing over and dosage effects, which modified the coloration of maize tissues specifically. The complex nature of the R locus has been confirmed and extended by molecular studies of Walker et al. (1995). Based on these and other studies, Stadler concluded that “Operationally, the gene can be defined as the smallest segment of the gene string that can be shown to be consistently associated with the occurrence of a specific genetic effect. It cannot be defined as a single molecule, because we have no experimental operations that can be applied in actual cases to determine whether or not a given gene is a single molecule.”

III. PHYSICAL CONCEPTS OF THE GENE Initially, the term gene coined by Wilhelm Johannsen (1909) referred to a hypothetical entity. Johannsen, one of the greatest authorities in genetics of that time, did not believe in the importance of chromosomes. By writing about the chromosome theory in 1914 he stated: “The present author does not accept the correctness of this hypothesis” and considered chromosomes as traits: “Man has 10 fingers and 32 teeth. One is a character just as much as the other.”

58

Re´dei et al.

By his death in 1927, he at least did not protest the use of the word gene to designate something concrete in the form of a “localized factor of inheritance” (Winge, 1958). William Bateson (1902), Johannsen’s contemporary and one of the most ardent propagators of Mendelism, coined the term allelomorph (Bateson and Saunders, 1902), which was later shortened to allele by Johannsen. In a posthumously published paper Bateson (1926) concluded: “I think we shall do to genetical science no disservice if we postpone acceptance of the chromosome theory in its many extensions and implications. . . . The hope that it may be safely extended into a comprehensive theory of heredity seems to me ill-founded.” A modern functional definition of the term genotype dates back to Woltereck (1909), who called it a reaction norm. The reaction norm referred to the range of phenotypic or expression potentials of a gene or genome. This concept essentially predicted that genes could permit a range of expressions leading to varying phenotypes depending on the genetic background, developmental, and tissue-specificity conditions, and different environmental stimuli. By contrast, Sturtevant and Beadle (1939) referred to the nature of the gene in a more subtle fashion by saying that “there is little in the way of facts to go on, and one is forced to resort to speculation. A reasonable supposition is that genes either are proteins or are associated with proteins.” Two decades later, George Beadle (1957) states: “The gene is defined as a localized unit of nucleic acid with a specific function, in higher forms closely associated with protein.” Even today, definitions of the gene found in textbooks and glossaries are often ambiguous. Generally, the term gene is defined operationally as DNA that “codes for a single polypeptide” (Klug and Cummings, 1983), or for “one particular product” (Elseth and Baumgardner, 1984), or as “DNA that codes for an RNA” (Pierce, 2003). According to Singer and Berg (1991) “a gene is a single hereditary unit.” They also note that “several different definitions are plausible but no single one is entirely satisfactory.” Snyder and Gerstein (2003) defined the gene as a “complete chromosomal segment responsible for making a product.” The latter authors list several functional attributes by mentioning that genes usually are open reading frames, which are characterized by constant sequences that may display codon bias, single nucleotide polymorphisms, or alternative splicing. They also note that genes could be overlapping or defective (i.e., pseudogenes). Concerning a precise definition of the gene, Dillon (2003) called recurrent attention to the position effects, which were originally discovered by Sturtevant (1925) during his studies of Bar duplications in Drosophila. These studies indicated that expression and regulation of a gene could be changed by physical alterations affecting its chromosomal neighborhood (Bridges, 1936). This was first illustrated genetically by the so-called “Dubinin-effect” showing that dominance of Drosophila wild-type H (hairy) allele over the mutant h allele

2. Changing Images of the Gene

59

was reduced by translocation of H from chromosome 3 into heterochromatin of chromosome 4 (Dubinin and Sidorov, 1934, 1935). These authors showed that removal of H from the translocated position restored dominance. Molecular basis of the “Dubinin-effect” was later subjected to detailed analysis revealing complex gene rearrangements accompanying the ci (cubitus interruptus) translocation (Locke and Tartof, 1994). Based on early genetic analyses of position effects Richard Goldschmidt’s (1958) suggested that genes in the chromosomes represent “hierarchial order of fields” controlled by specific regulatory cues. Stadler (1954) also referred to the contention of Goldschmidt (1958), who expressed a rather heretical view of the gene by stating that “There is no longer a gene molecule, but a definite molecular pattern in a definite section of the chromosome. . .” . . .“In this sense there is no normal gene.” This “Goldschmidtian idea” was dismissed (Sturtevant, 1965) or completely ignored (Demerec, 1955, 1967) because overwhelming evidence indicated that genes are genetically localized functional units along the chromosome (Sturtevant, 1913) that can be mapped by recombination, and their expression can be studied biochemically (Beadle, 1945).

IV. CHEMICAL NATURE OF THE GENE A. Uncertain substance of the hereditary material The first, investigator to develop a most elaborate, but naive and nebulous molecular theory of heredity, was the botany professor Na¨geli (1884). He suggested that in the cell of an albuminous plasma substance, a stereoplasm, pervades the inner volume enclosing also the genetic material, the idioplasm (p. 23). The stereoplasm and idoplasm would form micellae in which only the idioplasm codes for the linearly expanding hereditary anlagen that might function in a yeast-like, fermentation manner (p. 48). The stereoplasm appears to have only a structural and nutritional role (trophoplasm). The idioplasm is supposed to determine the trophoplasm (p. 47). “The activity of the idioplasm makes itself evident where heritable process of growth or metamorphosis takes place” (p. 29). “The idioplasma-net generates probably the frequently recurring net-like. . . structure of the nuclear substance (p. 41).” “The different form, size, and arrangement . . . of the idioplasm” (p. 26) account for the numberless differences and combinations in chemical and morphological processes. Unfortunately, Na¨geli failed to understand Mendel’s work (Na¨geli, 1867) and either did not know about the work of the chemist Miescher (1871) or he purposefully ignored it just as he ignored phenomena involving heredity in bacteria (Migula, 1897). On the contrary, Na¨geli’s contemporary, Oscar Hertwig (1875) recognized that the chromatin substance is universal in

60

Re´dei et al.

all organisms. Yet, this idea had minimal impact on the evolving theory of Mendelian heredity. Real milestones toward identification of the chemical nature of genetic material were reported only much later by Avery et al. (1944), who showed that the transforming principle of Griffith (1928) was DNA, and by Hotchkiss (1957), who proved that it was only DNA. According to Evelyn Witkin (2002), the discovery of significance of these publications, as well as those of Hershey and Chase (1952), and Watson and Crick (1953a,b), were not immediate: “Although Avery et al. . . . had demonstrated in 1944 that the genetic material is DNA, the prevailing attitude at Cold Spring Harbor had been respectful skepticism. Some suggested that the transforming DNA in their experiments had activated genetic information already present or somehow caused a directed mutation. Others believed that the minuscule trace of protein still contaminating the DNA was the active agent. Delbru¨ck declared DNA to be a stupid molecule, incapable of carrying genetic information.” Judson (1979, p. 59) reports an interview with Delbru¨ck, who similarly expressed his skepticism about the results of Avery’s laboratory: “. . . at that time it was believed that DNA was a stupid substance, a tetranucleotide which couldn’t do anything specific. So, one of those premises had to be wrong. Either DNA was not a stupid molecule, or—the thing that did the transformation was not the DNA.” To cast doubt on Avery’s and Hotchkiss’ results, Mirsky and Pollister (1946) suggested that even the purest DNA preparations could contain 1–2% protein. (Mirsky and Pollister used the Millon test for sensitive detection of protein levels. This procedure was developed in the nineteenth century by the Frenchman August, Nicolas Euge`ne Millon, based on the fact that mercury–nitric acid solution stained hydroxyphenyl groups and proteins red). Therefore, they concluded: “it is not yet known which the transforming agent is—nucleic acid or nucleoprotein.” Many arguments questioning the “faculty” of DNA as genetic material were based on a hypothesis of Levene and Lawrence (1931). This hypothesis suggested that nucleic acids have repeated units of equal numbers of four bases (A, T/U, G, and C) and therefore lack the necessary variation required for the genetic material. Levene’s hypothesis thus predicted a dull nature of nucleic acids, apparently supporting Shro¨dinger’s “periodic crystal” model. In fact, many experimental measurements available at that time indicated that in different samples of total cellular RNAs the four bases were not very different in amounts. The wrongheaded inference that nucleic acids consisted of monotonously repeating “units of 4” therefore suggested that DNA could not provide the specificity required for the variety of functions that different genes perform (i.e., because any given segment of a DNA macromolecule would qualitatively posses the same informational content as another segment). By contrast, proteins favored since the time of Na¨geli (1884) as potential carriers of genetic information were known to vary a great deal. That these types of molecules

2. Changing Images of the Gene

61

represented hereditary material were therefore the best possible idea under the circumstances of the available biochemical technology. A revolutionary change in this view happened in 1946, when Chargaff and associates conducted their studies of nucleic acids. Discovery of paper chromatography facilitated the quantitative analysis of minute amounts of organic molecules and Beckman developed photoelectric quartz spectrophotometers for commercial analytical measurements (Chargaff, 1979). Thus, it became feasible for Chargaff’s group to prove that base composition of DNA varies in different species but not among tissues of the same species (Chargaff et al., 1949). These new findings confirmed the view of Avery et al. (1944) that the genetic material—the “transforming principle”—was indeed DNA.

B. The DNA-double helix The hypothesis that DNA may serve as template during replication for the assembly of “protein genes” prompted intensive research on the structure of DNA, essentially commencing in the late 1940s (Butler, 1952). In February 1953, Pauling and Corey proposed a three-stranded DNA structure, which could however be hardly correlated with binomial segregation of Mendelian traits. Two months later, on April 25, 1953, Watson and Crick reported a double-helix model of DNA, which eventually spurred a dramatic impact on biology (Watson and Crick, 1953a,b). This model provided a simple molecular structure of the genetic material and opened a new approach for experimental study of most essential attributes of the gene—replication, mutation, and heterocatalytic function (i.e., expression of the DNA’s informational content into organismal phenotypes). The double-helix model allowed for a great variety of nucleotide sequences to be present along a given stretch of DNA and, at the same time, preserved a universal physical structure (such as base density along the track, general left turn of the helix, constant diameter among the diverse molecules, and regular pairing of the bases). Recognition that the double-helix model was a groundbreaking discovery took some time. According to anecdotal sources, H. J. Muller, one of the most prominent geneticists at that time, was on a Hawaiian vacation and missed the April issue of Nature. He caught up with reading only a couple of months later (Carlson, 1981). Other leading geneticists (Stadler, 1954) did not immediately or not entirely recognize the significance of Watson and Crick’s model. Interestingly, even Watson expressed some doubts in a letter written to Delbru¨ck on March 22, 1953. “I have a rather strange feeling about our DNA structure. If it is correct, we should obviously follow it up at a rapid rate. On the other hand it

Re´dei et al.

62

will at the same time be difficult to avoid the desire to forget completely about nucleic acid and to concentrate on other aspects of life” (Judson, 1979; p. 229).

C. RNA genes Although Albrecht Kossel discovered RNA before the end of the nineteenth century (Hammarsten, 1894), the recognition of its genetic significance was long delayed. Textbook definitions of the gene often gloss over RNA, the other carrier of genetic information. The second chapter in Lewin’s (2004) textbook is “The Gene is DNA.” In 1955, Fraenkel-Conrat and Williams showed that, upon packaging into empty protein coat of tobacco mosaic virus (TMV) the RNA of closely related Holmes Ribgrass virus, the newly formed viral particles expressed the genes of the RNA donor rather then the protein coat. This clearly demonstrated that the genetic material of these viruses must be RNA. Subsequently, Gierer and Mundry (1958) used nitrous acid (HNO2) as a chemical mutagen to alter the RNA genes of TMV. This compound oxidatively deaminates cytosine into uracil, and adenine into hypoxanthine. The latter indirectly results in an A ! G transition. After such treatments mutant virus and mutant RNA were produced. Guanine was converted into xanthine, which interfered with replication of the virus. Somewhat later, it also turned out that RNA is not just a crucial molecule for protein synthesis, but it can also be reverse-transcribed into DNA (Baltimore, 1970; Temin, 1964), mandating a revision of the “central genetic dogma” regarding information flow from DNA through RNA to proteins (Crick, 1958). One of the most significant discoveries was the recognition of regulatory roles of noncoding antisense and short interfering RNAs (RNAi) (Fire, 1999; Hutva´gner and Zamore, 2002) in the control of expression of DNA genes. The literature generally distinguishes microRNAs (miRNA) and short RNAi, which are generated by cleavage of longer precursor transcripts by the Dicer ribonuclease, and further processed into 21–22 (19–23) nucleotide RNAs by RNAinduced silencing complex (RISC; Bartel, 2004; He and Hannon, 2004; see also Insight Reviews (2004). Nature 431, 338–378). These short RNAs are considered medically important because of specificity for preventing undesirable gene function. Lately reservations were raised, however, because of their effects on unintended targets (Jackson and Linsley, 2004). One of the first examples for regulatory roles of small RNAs was provided by two highly specific miRNA genes (mir-136 and mir-127) in the mouse chromosome 12 (corresponding to human chromosome 14q32). These genes are expressed in the maternal chromosome and act as antisense to the retroposon-like gene (Rtl1), which is expressed only from the paternal chromosome (Seitz et al., 2003; see discussion of imprinting in a later section).

2. Changing Images of the Gene

63

Numerous expression vectors were designed for gene silencing in a high-throughput manner (Zheng, 2004). For example, small RNAs libraries for 9610 human and 5563 mouse genes are available for the analysis of gene functions by silencing (Paddison et al., 2004). There are additional cases of noncoding RNAs, which play roles in mediating the binding of transcription factors and thus regulate gene expression (Cawley et al., 2004). For example, a noncoding RNA transcribed from an intergenic region upstream from the SER3 gene in yeast interferes with binding of activators to the SER promoter, and thus regulates transcription (Martens et al., 2004).

V. THE GENETIC CODE One of the early proposals on how a chemical substance could code for a wide array of biochemically meaningful functions was made by Oscar Hertwig. In his book, “The Cell” (originally published in German in 1893), he wrote: “the hypothetical idioblasts . . . are according to their different composition, the bearers of different properties, and produce, by direct action or by various methods of cooperation, the countless morphological and physiological phenomena, which we perceive in the organic world. Metaphorically they can be compared to the letters of the alphabet, which though small in number, when combined form words, which in turn, combine to form sentences or to sounds, which produce endless harmonies by their periodic sequence and simultaneous combination.” (Hertwig, 1895, p. 349) Friedrich Miescher suggested a similar genetic alphabet in a letter to his uncle, the embryologist Wilhelm His (Olby and Posner, 1967). Chargaff (1955) expressed a pessimistic view about the chances of decoding the manner by which genetic molecules are read out into cellular constituents: “I believe . . . that while the nucleic acids, owing to the enormous number of possible sequential isomers, could contain enough codescripts to provide a universe with information, attempts to break the communications code of the cell are doomed to failure at the very incomplete stage of our knowledge. Unless we are able to separate and to discriminate, we may find ourselves in the position of a man who taps all the wires of a telephone system simultaneously. It is, moreover, my impression that the present search for templates, in its extreme

Re´dei et al.

64

mechanomorphism, may well look childish in the future and that it may be wrong to consider the mechanisms through which inheritable characteristics are transmitted or those through which the cell repeats itself as proceeding in one direction only.” Nevertheless, a mere 5 years later, the laboratories of Nirenberg and Ochoa reported independently the first experimental decoding in 1960. Only about 5 years later, the job was completed (for review see Ycˇas, 1969). The “magic number” suggestion of 64 (43) triplets of 4 bases coding for 20 natural amino acids (sliding over the matter of nonsense codons for the moment) has withstood the test of time reasonably well. However, the originally assumed universality of the code from Escherichia coli to amphibians and mammals (Marshall et al., 1967) required some revisions (Barrell et al., 1979), as the same codons have somewhat different meaning in some organisms. For example, in the small genome of Mycoplasma capricolum the supposedly universal UGA stop codon can code for tryptophan, whereas the standard UAA stop codon in the ciliated protozoa Tetrahymena thermophila codes for glutamine. Especially, within mitochondria of different fungi, Drosophila and mammals there are a variety of triplets that do not conform to the standard code. In some organisms, in addition to the AUG initiator codon, AUA, AUU, and AUC may also start translation as methionine codons. On the other hand, some mitochondrial genes have no stop codons at the end of their reading frames. In such cases, U or UA terminate the transcripts and, after processing, this sequence may become a UAA stop signal by addition of A/AA (Knight et al., 2001). The UGA codon in certain animal genes encodes the 21st amino acid, selenocysteine (Korotkov et al., 2002), whereas UAG in Archaea and Eubacteria may code for the 22nd amino acid, pyrrolysine (Hao et al., 2002). Intriguingly, programmable ribozymes can also attach nonnatural amino acids to tRNAs and incorporate them into proteins (Bessho et al., 2002).

VI. STRUCTURE OF THE GENE A. Step allelomorphism and pseudoalleles Classic presentations of the chromosome, as a string of beads (chromomeres) and that of the genes, as the units of function, recombination, and mutation (Morgan et al., 1915), had been challenged by the discovery of step allelomorphism (Agol, 1929; Dubinin, 1929) and pseudoallelism (Hogness et al., 1985; Lewis, 1945; Oliver, 1940). The allelism test is a classically genetic way of defining the gene as a functional unit. If two recessive genes are allelic, they fail to complement each other in the F1 hybrids (i.e., the hybrid has mutant

2. Changing Images of the Gene

65

phenotype). In case the hybrid of two recessive individuals is of wild phenotype (i.e., the mutations complement each other), the two genes are not allelic. Thus, the number of complementation groups reveals the number of different loci. In the late 1920s, Russian laboratories worked with various scute (sc; now called achaete-scute complex, ASC) alleles, which are involved in the determination of macro- plus microchaetae, and bristles in Drosophila. They observed that various combinations of different sc alleles (some of which actually correspond to chromosomal rearrangements) led to stepwise pattern changes on the fly body. To illustrate this case, let us designate sc alleles for four different patterns with numbers 1, 2, 3, 4. (Alleles designated by 0 do not express, while the others positively mediated a phenotypic effect.) According to these researchers, upon crossing a female carrying alleles 1, 0, 0, 4 with a male harboring alleles 0, 0, 3, 4, the diploid F1 hybrid shows the absence of sensilla (sensory organs) determined by allele 2. This is because allele 2 is silent in both parents and their F1 hybrid, which has a genotype 1, 0, 0, 4/0, 0, 3, 4 (the missing expression at site 2 is marked by bold 0). The lacking expression of other sc alleles will display different patterns, resulting in step-like alteration of the phenotype. This characteristic anomaly suggested to Agol, Dubinin, and colleagues that there are subgenes; so a genetic locus as conventionally defined would not be the ultimate unit of heredity. This was inconsistent with the then prevailing idea that the gene locus is the ultimate unit of function and that the alleles would be stereochemical modifications of an indivisible molecule, as envisioned by the early Morgan school. Actually, step allelomorphism proved to be a specific form of partial, allelic complementation (Catcheside, 1960). Discoveries revolving around pseudoallelism seemed to violate further the unitary concept of the gene. In this phenomenon, certain alleles are not just partially complementary, but recombination events between the sites of such gene can also occur. Alleles displaying such properties were considered pseudo (i.e., nongenuine) because, according to the prevailing theory, recombination within a gene (i.e., a crossover event occurring between the sites of two allelic mutations) was not supposed to occur. In retrospect, there was no good reason to suppose that arbitrary choices for recombination sites, made by the nuclear machinery that mediates this process, would be unable to choose a stretch of DNA that happens to fall between the sites of two mutations located within a given gene. The possibility that recombination could occur between alleles of a gene had been predicted earlier by Offermann (1935), a cytogeneticist colleague of H. J. Muller. He assumed that some genes flanking the alleles of a studied target gene could exert a regulatory position effect on the target gene, even if they do not confer themselves any other detectable phenotype. Recombination events separating such a target gene from the cis-acting regulatory factor would thus give the impression that the gene is divisible, because recombination can

66

Re´dei et al.

Figure 2.1. Pseudoallelism. The a1 and a2 mutations in a gene are pseudoallelic (i.e., there is no interallelic complementation between a1 and a2). In trans-arrangement (at left) they confer mutant phenotype. Crossing-over between a1 and a2 occurring at low frequency results in two recombination products with exchanged flanking markers. One chromosome (upper) carries the two recessive alleles (a1 and a2) in the same strand, conferring mutant phenotype, whereas the lower strand carries a wild type alleles (þ, þ) because of recombination between the two sites of the gene. The flanking markers (Y/y and Z/z) indicate that appearance of the wild phenotype did not result from back mutation, but from crossing-over.

remove the position effect. The first convincing evidence for genuine intragenic recombination was derived from the study of Drosophila’s lozenge (lz) locus by Oliver (1940). Among the progeny of females heterozygous for lzs and lz9, Oliver found wild type flies appearing at a frequency of 10–3, which displayed an exchange of flanking markers of the lzs and lz9 alleles. Thus, the rare exceptional progeny seemed to occur by crossing over and not by mutation (see Fig. 2.1). Pseudoallelism was another argument for the revelation that the gene was no longer the unit of mutation and recombination. A gene could formally be “broken down” by recombination (irrespective of any molecular knowledge about the locus and its mutations). This need not have caused anyone to perceive as the demise of the “gene concept” in general, because there was no compelling argument in favor of viewing a locus as being merely a “point”—as opposed to reality, that any and all genes have linear dimensions. However, the changing view of the gene—as exemplified by the phenomena just described— was, however, historically significant.

B. Transposable elements The “brilliant investigations” (Stadler, 1954) of Barbara McClintock opened an entirely new view on the organization of genetic material. She showed that broken chromosome ends can fuse (McClintock, 1942). These early studies led to the discovery of “controlling elements” in maize (McClintock, 1956) and prompted subsequent revelations about the insertion elements (IE) and transposons of prokaryotes (Cohen, 1976; Jordan et al., 1968). It was an unexpected discovery that certain genes do not just function at a fixed position but can move

2. Changing Images of the Gene

67

from one place to another and integrate into other genes by the function of transposase enzyme(s). This discovery was fundamentally different from observations involving mechanical translocation, inversion, and transposition of chromosomal segments (Bridges, 1923; Sturtevant, 1926). Eventually, information on mobile genes was summarized in an approximately 1000-page tome (Berg and Howe, 1989). Transposable elements display a great diversity, but the active elements share one common feature of having transposase gene(s). The IS may have only the transposase function and can transfer other genes only if they are associated with IS. Other elements are much longer, can accommodate several genes, and can mediate their own movement, as well as the transposition of other elements (Craig et al., 2002). The study of transposable elements revealed a new aspect of genetic evolution, whereby lateral/horizontal transfer of genes among diverse organisms, as has occurred during evolution (Boucher et al., 2003; Miller and Capy, 2004). Here is an example: the genetic material of eukaryotes is sequestered into nuclear and mitochondrial compartments and also within plastids in plants. These compartments seem to have interchanged genes during evolution (Thorsness and Weber, 1996), involving potential transposition events. For example, in the 2nd chromosome of Arabidopsis 135 genes appear to be of plastid origin; whereas 618 kb of mitochondrial DNA was found in the centromeric region, part of which is actively transcribed (Stupar et al., 2001).

C. Nonsexual transfer of genes Discovery of restriction endonucleases and DNA ligases during 1968–1972 opened the feasibility for genetic engineering in prokaryotes (Cohen et al., 1972; Jackson et al., 1972). This feat could be readily applied within a decade to mammalian cells (Capecchi, 1980) and, with the agrobacterial Ti plasmid vectors, also to higher plants (De Greve et al., 1982; Herrera-Estrella et al., 1983). Later, a battery of powerful new reporter genes encoding various selectable markers, luciferases (Koncz et al., 1987; Schneider et al., 2000), betaglucuronidase (GUS; Jefferson et al., 1987), and green fluorescent proteins (GFP; Tsien, 1998) opened the way for monitoring gene activity in living organisms. These markers also facilitated tracking a given transferred gene. Furthermore, the gene gun method (Klein et al., 1987) facilitated transformation of important crops, and many other species of plants and animals, which earlier were not amenable to this type of gene transfer. The growing literature of genetic transformation in eukaryotes necessitated a new term for the introduced genetic factors as “transgenes” (Gordon and Ruddle, 1981). In summary, the wherewithal to mark and transform or transfect “DNA of interest” has revolutionized molecular analyses of cellular and developmental phenomena. As a

68

Re´dei et al.

consequence, many puzzling questions of classic genetics could be answered using the extraordinary resolving power that has arisen, thanks to the application of novel molecular principles and the tools, which followed quickly from their conceptualization.

VII. MOLECULAR UNITS OF THE GENE Changes in the definition of a gene as an intact recombinational, mutational, and functional unit have happened gradually and involved heated arguments and discussions (Carlson, 1966). The terms recon, muton, and cistron were introduced as new definitions of the genic units during the 1950s (Benzer, 1957). Recons and mutons turned out to be single nucleotides. In this regard, Yanofsky (1963) studied recombination within gene A of E. coli’s tryptophan operon. He found that a CCT codon at position 211 of gene A spells glycine, but this site was altered to GCT (arginine) and CAT (valine) in two mutants, respectively. Using appropriate flanking markers in phage-mediated transduction experiments, recombination was detected between nucleotides of GCT and CAT codons, which “restored” the wild-type CCT glycine codon. This experiment provided evidence that recombination can take place between nucleotides. The term cistron was originally defined as the unit of molecular function, specifying the amino acid sequence of a polypeptide. A cistron was tacitly assumed to be an uninterrupted unit. This classic definition was challenged by independent investigations on adenovirus-2, which pointed to the fact that many mRNAs are mosaics of spliced sequences that are transcribed from multiple physically separated tracts of DNA (Berget et al., 1977; Broker et al., 1977). By providing a coherent model, Gilbert (1978) summarized several experimental evidences indicating that genes may be “in pieces,” such that a given transcription unit commonly consists of translated exons and apparently noncoding introns. In fact, it was found later that in eukaryotic genes the number of exons varies from one to up to 236 detected in the huge Titin gene, which encodes a protein controlling fiber network in skeletal muscles (Gerull et al., 2002). Alternative splicing of primary transcripts, providing different combinations of exonic sequences, might generate multiple mRNAs for different proteins. For example, the immunoglobulin genes are well known for generation of thousands of antibodies, and a large variety of T-cell receptors result from a much more limited number of genes (Honjo et al., 1981; Wang et al., 2002). This phenomenon is also called exon-shuffling, which stands for generation of different proteins by alternative splicing of transcripts—and has also geneevolution consequences (Patthy, 1999). Various studies estimate that 40–60% of the human genes are alternatively spliced. This phenomenon is also common in rodent genomes (Modrek and Lee, 2003). Overall, 10–30% of alternatively

2. Changing Images of the Gene

69

spliced human genes produce tissue-specific splice forms. Alternative splicing is thus a common means of differentiation, particularly in the brain, and in the immune system (Xu et al., 2002). Alternative splicing may also explain why humans can fulfill many more life functions with fewer genes (25,000–30,000): International Human Genome Sequencing Consortium, 2004) than, for example rice plants (50,000–60,000: Feng et al., 2002; Sasaki et al., 2002). Alternative splicing is often coupled to alternative promoter usage, which is regulated by various tissue-specific and environmental stimuli (Schibler and Sierra, 1987). One of the first examples for alternative promoter usage came from studies of transcription of different yeast invertase genes (Carlson and Botstein, 1982). Subsequently, two different myosin light-chain proteins were found to derive from the same gene in mammals, due to differences in transcription initiation and splicing (Periasamy et al., 1984). In a more complex case involving the human interleukin-4 gene, at least 13 regulatory sites were found to respond to specific promoter elements (Guo et al., 2001). These cases clearly show that the applicability of classic “one gene–one polypeptide” concept (Beadle, 1945) is a rather antiquated notion, especially in eukaryotes. DNA sequence units comprising exons often define distinct intrapolypeptide domains. Domains are defined as segments of proteins with characteristic tertiary structure that fold independently and usually define specific functional properties. Similar domains may be shared by different proteins performing completely unrelated functions (Coin et al., 2003). Thus, the function of a given gene product is evidently not a simple sum of its individual domain components. Nevertheless, according to the “Rosetta Stone” hypothesis of Marcotte et al. (1999), comparative analysis of domains (i.e., reflecting conservation of exon sequences) provides a useful tool to study evolution and regulatory significance of protein interactions (e.g., in the context of functional analysis of multiprotein complexes). An evident evolutionary advantage of modular domain organization is that mutations affecting a specific domain may alter only one specific feature (e.g., a domain interaction required for proper intracellular targeting) without fully destroying the function of a protein (e.g., enzymatic activity; Bar-Joseph et al., 2003). In contrast to coding sequences of exons, introns were initially called “selfish” (Orgel et al., 1980) and “parasitic” (Orgel and Crick, 1980) and—as spacers, pseudogenes, and other common repetitive sequences—were considered evolutionary baggage of junk. One of the first indications that introns may have functions was derived from studies of the adjacent mitochondrial cytochrome oxidase (oxi3) and apocytochrome b (cob) genes in yeast. The box 7 intron proved to be essential for processing the oxi3 transcript, whereas box 3 coded for a maturase of cob transcripts (Lazowska et al., 1980). These results showed that introns are not absolutely indispensable elements. The intron-less, short genes functioned well without them.

70

Re´dei et al.

By the mid-1990s several other experiments indicated that introns perform important regulatory functions. Thus, certain introns were found to enhance the expression of transgenes (Choi et al., 1991), to promote the accumulation of transcripts, and to enhance translation (Balakirev and Ayala, 2003; Nott et al., 2003). Stated another way, it is not uncommon to find enhancer elements within intronic sequences, in addition to being located in their usual positions upstream or downstream of a gene’s coding region. Pseudogenes, which like introns are typically untranslatable because they contain stop codons and other mutations, were also found to regulate stability of their homologous counterparts in mice (Hirotsune et al., 2003), as well as in plants (Yamada et al., 2003). Our views about the highly compacted and then presumed functionally silent heterochromatin have also changed considerably. Earlier, it was assumed that the few scattered genes in the heterochromatin might actually represent a kind of short interspersed euchromatin. Until better nucleotide sequencing became available for repetitive heterochromatic regions, this problem could not be resolved in a satisfactory manner. Today, we know that heterochromatin is not quite idle, but it may show transcription from both DNA strands (Yamada et al., 2003). Compacted stretches of DNA carrying repetitive sequences appeared genetically silent. Yet, embedded in heterochromatin there are a few bona fide transcription units. Paramount examples come from Y chromosomes in animals, which are largely devoid of alleles densely distributed along their homologs (the X chromosomes). The presence or absence of Y has clear phenotypic meaning, even when this chromosome is not sex-determining. Thus, for example, the Y chromosome of D. melanogaster shares alleles with only a few X-chromosomal factors, but this Y also contains seven genes, whose expression is necessary for male fertility (Carvalho, 2002; Meller and Kuroda, 2002).

VIII. HIGHER ORDERS OF GENES After the discoveries of coordinated regulation of two or more genes—exemplified by the tryptophan gene cluster of Salmonella (Demerec and Demerec, 1956)—a short time was required to reach the operon concept (Jacob and Monod, 1961). In a way, these phenomena led eventually to considerations of genetic networks, and even to studies of the proteome (Date and Marcotte, 2003; Gavin et al., 2002; Giot et al., 2003; Kamp et al., 2004; Maslov and Sneppen, 2002; Zhu et al., 2003). Genome-wide analysis of gene expression has led to introduction of the term “supraoperon,” which refers to clusters of individual operons that show similar transcriptional regulation. Bacterial supraoperons may include 90 or more genes, which also show functional clustering (i.e., in a common metabolic or regulatory pathway; Berka et al., 2003). In

2. Changing Images of the Gene

71

eukaryotes it is also common that genes acting in related metabolic or developmental processes are expressed coordinately (Maslov and Sneppen, 2002). During recent years several powerful molecular and bioinformatics tools became available for genome-wide analysis of temporal and spatial regulation of gene expression. Hartwell et al. (1999) pointed out the timeliness of investigating the correlation of these data with interaction maps of corresponding gene products. Development of yeast two-hybrid technologies for monitoring physical interactions between gene products (Brent et al., 1997; Fields and Song, 1989; McAlister-Henn et al., 1999) allowed detection of functional networks of thousands of proteins (Gavin et al., 2002; Maslov and Sneppen, 2002). The two-hybrid approach implies that if the function of one protein is known, the function of its associated partners may also be inferred. In addition, cross-species comparisons of protein-interaction maps, when handled with necessary caution, offer useful hints about possible functions of unknown proteins. Such entities can be revealed to share common domains with well-characterized proteins from other species. Moreover, similar regulation of genes that code for interacting proteins helps to define coherent networks of coregulated genes and proteins that act in common or interlinked pathways. Based on the huge amount of two-hybrid data available, 3875 functional linkages of 804 proteins were confirmed in yeast by Date and Marcotte (2003). A draft Drosophila protein-interaction map for 7048 proteins with 20,405 interactions was reported by a computational method of rating the available twohybrid interaction data, and at higher confidence confirmed 4780 interactions among 4679 proteins (Giot et al., 2003). The network showed a short-range organization, presumably corresponding to multiprotein complexes, and a more global organization of intercomplex connections (Giot et al., 2003). The results of these protein-interaction studies can now be confirmed by mass spectrometry analysis of protein complexes (Zhu et al., 2003). In addition, several computational methods can be applied to examine the coinheritance of functional linkages, even across phylogenetic boundaries (Yandell and Majoros, 2002). In bioinformatics terms, central elements of interaction networks are called nodes, which are characterized by their degree of connectivity (i.e., how many links a node has to other nodes). The highly connected nodes are called hubs. Most of the biological networks are scale-free networks because their degree of distribution approximates a power law (i.e., P(k) k– ). Small increases the role of a hub, whereas larger than 3 indicates irrelevance of a hub. When ¼ 2, a hub-and-spoke network appears and the largest hub is linked to a large fraction of nodes (Baraba´si and Oltvai, 2004). Biological systems interact in a nonrandom, scale-free manner, reflecting the existence of special functional hubs (Baraba´si and Albert, 1999). The path length indicates the number of links between two separated nodes. Metabolic networks, in which substrates and products determine a direction of flow, represent directed networks.

72

Re´dei et al.

By contrast, protein interactions detected by two-hybrid studies also include undirected networks representing mutual interactions. Nonetheless, even these undirected networks can display directed processes, such as those involved in signal transduction. When one node is connected to several others to mediate, for example, signaling interactions, these interactions can be characterized mathematically by the clustering coefficient. At higher level, clusters may form different types of hierarchial structures, revealing possible cross talk and regulatory connections between various pathways (Maslov and Sneppen, 2002; Ravasz et al., 2002). Computational modeling is not only applicable to the analysis of protein-interaction networks but also to modeling temporal and spatial regulation of transcription, and to studying regulatory interactions between genes acting in common or different pathways (Stolc et al., 2004). It has been concluded “the genomes exquisite control of each gene’s activity—and not the genes per se—that matters most” (Pennisi, 2004). When a network cluster mediates unidirectional information flow, representing an ideal linear signaling pathway, the order of nodes or modules used in that pathway can be easily dissected genetically by the analysis of epistasis. Historically, the term “epistasis” refers to the obvious concept that development of a particular phenotype is usually the result of cooperative effects of more than one gene at different loci. Thus, in a linear pathway controlling a given response, the sequential order of elements can be determined by classic genetic analysis of phenotypes of double mutants. However, if a function represents a potential hub (i.e., a node protein involved in multiple regulatory interactions), mutations of the corresponding gene may affect multiple cellular functions, resulting in alteration of several phenotypic traits. Historically, mutations affecting multiple traits—often both quantitative and qualitative ones—were termed pleiotropic. This term was coined by Ludwig Plate (1913) to encompass phenomena, in which single genes exhibit multiple effects. Cross talk between diverse regulatory pathways provides ample examples for pleiotropic functions. Thus, many elements of different signal transduction pathways (e.g., protein kinases, phosphatases, transcription factors, proteasome, and signalosome components) have been shown to affect either expression of multiple genes, or functions (i.e., cellular localization, activation, or stability) of multiple proteins, or both. Mutational alterations of corresponding genes therefore result in pleiotropic defects (Collins et al., 2003). As a single pleiotropic regulatory gene may control multiple quantitative traits, it is difficult to determine how many of the identified quantitative trait loci (QTLs) have or result from pleiotropic effects due to mutational alteration of multifunctional regulatory proteins (Brockmann et al., 2000; Ferrall, 2004; Moraes, 1998; Varona et al., 2004). Various examples of regulation also illustrate that biological systems are characterized by robustness (Albert et al., 2000). This means, that despite their complexities, the mutational inactivation of some

2. Changing Images of the Gene

73

nodes does not necessarily lead to total breakdown, although perturbation of major hubs may have more severe consequences. The pleiotropic nature of regulatory proteins can be exploited experimentally. The transcription-associated factors (TAF) by virtue of their different domains can modulate transcription of different genes. For example, randomized libraries of zinc-finger containing artificial transcription factors can induce phenotypic variations in yeast and mammalian cells. By linking multiple zincfinger domains together with diverse DNA-binding specificities, new transcription factors were generated. When each was fused to either a transcription activation or repression domains, the transfected cells displayed diverse phenotypic changes, such as drug resistance, thermotolerance, or osmotolerance in yeast, and neuronal differentiation in mouse neuroblastoma cells (Park et al., 2003). Developments discussed in an earlier section illustrate that modern integrative molecular biology looks at genes not just individually and separately, but it seeks out their systems biology (Hood and Galas, 2003). The term systems biology thus stands for global study of biological mechanisms by monitoring gene, protein, and informational pathways by using efficient bioinformatics methods to develop rational models for understanding complex controlling systems of cells, tissues, and whole organisms (Ideker et al., 2002; Weston and Hood 2004; Xia et al., 2004; Yandell and Majoros, 2002). Studying interactive systems of genes goes far beyond basic biological interest because it promises formerly unimaginable changes for predictive and preventive medicine (Hood et al., 2004).

IX. GENE NUMBER AND GENE SIZE After sequencing of numerous prokaryotic and eukaryotic genomes, the concerns of classic genetics with gene size and gene number retain historical importance. The number of discrete genetic factors possessed by a given organism was of interest since the time of Na¨geli (1884), who speculated that trees might have billions of hereditary units. For the first time, using three independent genetic and cytological methods, the minimal numbers of Drosophila genes were estimated as ranging from 1150, 1425, to 1800 by Muller (1929). Somewhat later, Gowen and Gay (1933) came up with a figure of not less than 14,380 genes for Drosophila by estimating the gene number on the basis of mutation rate at specific loci and overall mutation frequency. The latter figure is remarkably close to the 13,600 genes indicated by the genome sequence data (Adams et al., 2000). A counter example is provided by the rather obscure estimate of Garcia-Bellido and Ripoll (1978), who speculated that the gene number of Drosophila is at least 5000, but not more than 80,000, based on the number

74

Re´dei et al.

of salivary chromosome bands, and the assumption that the majority of bands contain 20,000 bp. The upper estimate was derived roughly from the fact that D. melanogaster salivary-gland chromosomes contain approximately 5200 bands, and that the average such entity might possess up to 20 genes, assuming that they code for proteins of modest size. (Although the existence of so many gene products with more than approximately 300 amino acids, and the existence of introns, could result in the estimate of “20 genes per band,” this guess remains a quaint piece of forgotten history). Due to evident uncertainty of their estimate, these authors concluded that “this evaluation does not directly tell us the number of mutable loci present in the genome; we depend for that on mutagenesis experiments.” In plants the number of genes was first estimated in Lilium pardalinum as 2193 by counting the number of chromomeres in the large pachytene chromosomes (Belling, 1928). Later, based on messenger RNA abundance, the gene number in the allotetraploid species Nicotiana tabacum was estimated to be 60,000 (Kamalay and Goldberg, 1980). Thus far, we have no exact data on the gene number for either Lilium or tobacco from genome sequencing. Nevertheless, based on genome sequence information from the rice plant (estimated gene number 46,022–55,615; Feng et al., 2002; Sasaki et al., 2002), and the dicotyledonous species Arabidopsis (estimated gene number 30,700; http://signal. salk.edu/cgi-bin/tdnaexpress), we can conclude that Belling’s figures for Lilium are too low, whereas the gene number for tobacco is likely closer to the expected value. However, one can more precisely evaluate a gene number estimate for Arabidopsis, which was obtained based on determination of the mutation rate and calculation from the zero class of the Poisson distribution (e–m) by Re´dei et al. (1984). These authors estimate of the gene number 28,750 in Arabidopsis. Based entirely on genetic data, is not much less than the most recent value of 30,700 (June 29, 2004) obtained after reannotation of the sequenced genome (TIGR Annotation Database). The size of genes was also estimated in diverse organisms before the recognition of DNA as the genetic material. Muller (1929) came to the conclusion that the size of Drosophila genes is in the range of 0.2–0.3 mm3, as speculated from cytological measurements of chromosomes. On the basis of limited DNA sequence information available at that time for prokaryotes, Demerec concluded in 1967 that the individual genes vary in size from 36 to 3500 nucleotide pairs. As we know today, the size of genes may vary from 15 nucleotides, encoding a 23S ribosomal subunit pentapeptide (Tenson et al., 1997), and the 21 bp of microcin C7 translational inhibitor of Enterobacteria (Gonza´lez-Pastor et al., 1994) to the human dystrophin of about 2.34 106 bp (Tennyson et al., 1995). However, the “average” size of processed genes (mRNAs) may be about 400 bp. Processed genes can also be viewed as reverse-transcribed from mRNA, thus free of introns and other noncoding sequences (Hollis et al., 1982).

2. Changing Images of the Gene

75

Of course, the average is not equal to the median and cannot represent the distribution of the entire range of organisms from prokaryotes to the highly evolved eukaryotes. The number of the eukaryotic coding nucleotides generally varies between 1100 and 1300 bp, but many genes have much longer coding sequences. Transcription units—exons, introns, 50 and 30 untranslated sequences combined—frequently occupy several dozen kilobases of genomic DNA in eukaryotes. According to an estimate, 26,564 annotated human genes show an average of 8.8 exons and 7.8 introns. About 80% of the human exons are less than 200 bp. Fewer than 0.01% of the introns are less than 20 bp, and fewer than 10% are longer than 11,000 bp (Sakharkar et al., 2004). As mentioned in an earlier section, a large fraction of human genes are alternatively spliced (Patel and Seitz, 2003), and thus the same gene may be translated into three or more variable protein isoforms. The size and number of a fraction of genes may show considerable variation even among closely related species, as new genes may originate by duplication, which could be followed by mutational alterations to gain new functions. Alternatively, new functions may arise by reorganization of exons, or acquisition of functional sequences from noncoding DNA tracts. As we have already mentioned, lateral/horizontal transfer can also deliver genes from one organism to another, leading to the evolution of new orthologs or paralogs with modified or new functions (Boucher et al., 2003). Alternative mechanisms, such as antibody gene switching (Honjo et al., 1981) or movement of transposable or insertion elements, can also significantly contribute to natural variations of sequence and size of genes in many species (Venter et al., 2001).

X. GENES IN THE CYTOPLASM The fact that there are traits that are unlikely to be in compliance with the Mendelian rules was first stated by Correns (1904): “Es scheint mir wenig Aussicht zu sein, die tatsachen den Mendel’schen Regeln in Einklanng zu bringen.” Correns (1909) and Baur (1909) provided definitive proof for plastid-controlled genetic traits (Hagemann, 2002). Nevertheless, Morgan (1926) advised geneticists: “... all known characters can be sufficiently accounted for by the presence of genes in the chromosomes. In a word the cytoplasm may be ignored genetically.” Preer (1963), an eminent contributor to infectious heredity of cytoplasmic particles of Paramecium, remarked: “Cytoplasmic inheritance is a little bit like politics and religion from several aspects. First of all, you have to have faith in it. Second, one is called upon occasionally to give his opinion of cytoplasmic inheritance and to tell how he feels about the subject.” Ten years later Curt Stern (1973; p. 172) came to the conclusion that “In man, there is

76

Re´dei et al.

no clear example of transmission of a trait that requires the assumption of extranuclear inheritance.” Today, functional importance of chloroplast and mitochondrial genes is evident in plants (Hagemann, 2002; Newton et al., 1996; Williams and Levings, 1992), and thousands of genetic examples underline an extraordinary importance of mitochondrial genes in mammals, as well as disease syndromes, aging, anthropology, and evolution of humans (Budowle et al., 2003; Harpending and Rogers, 2000; Moraes, 1998). The size of the mitochondrial genome is 366,924 bp in Arabidopsis, but many plants (e.g., cucumber) carry larger mtDNAs. In comparison, mtDNA of mammals is very small (16.5 103 bp) and contains only 37 genes. Nonetheless, this organelle harbors about 1000 proteins; most of which represent imported products of nuclear genes. Therefore, most proteins encoded by mtDNA are found together with nuclearly encoded subunits of mitochondrial protein complexes. Similarly, the majority of proteins encoded by chloroplast DNA in plants are combined in various complexes with nuclearly encoded proteins, many of which are imported into chloroplasts in a lightdependent fashion. One of the best known examples is the chloroplast DNA encoded large subunit of ribulose bisphosphate dicarboxylase (Rubisco) protein, which forms a complex with the nuclearly encoded small subunit of Rubisco (Chen et al., 1975). There are ample examples for genetic studies of mutations affecting chloroplast genes that cause various deficiencies in chlorophyll biosynthesis, photosynthesis, amino acid, and hormone metabolisms, or chloroplast DNA replication. Historical contributions to chloroplast genetics are discussed in depth by several reviews (Birky, 2001; Leister and Schneider, 2003; Surpin et al., 2002). Genetic dissection of mitochondrial gene functions, especially in mammals and humans, became fully feasible after development of appropriate molecular tools. The major practical problem to overcome was heteroplasmy, meaning that more than a single type of mitochondria occurs in the cell/body. As females often irregularly transmit mixed mtDNA molecules, genetic identification of mtDNA mutations (occurring at a frequency 10–3) is difficult by conventional genetics means. For example, in mice three nuclear QTL loci appear to affect mitochondrial sorting out (i.e., the segregation of genetically different mitochondria into different cell lines; Battersby et al., 2003). Molecular studies indicated early on that numerous human disease syndromes (e.g., hypotonia [reduced muscle tension], ptosis [drooping down eyelids], ophthalmoplegia [eye muscle paralysis], or high level of lactate in the blood serum, and so on) are associated with either mutations or reduced levels of mtDNA (Lestienne, 1999; Moraes, 1998). Online Mendelian inheritance in man (OMIM) (www.ncbi.nlm.nih.gov/) lists 348 human diseases relevant to mitochondria. Because subunits of mitochondrial protein complexes are also encoded by nuclear genes, these mitochondrial diseases have counterpart syndromes

2. Changing Images of the Gene

77

controlled by nuclear factors. For example, ophthalmoplegia is also part of more than 100 hereditary diseases controlled by different autosomal and X-chromosomal loci (see OMIM). Therefore, it is obvious that mutational effects of mitochondrial genes are often pleiotropic and also affect the symptoms of several nuclear mutations in a complex manner. The majority of mitochondrial diseases are associated with alterations in the mitochondrial respiratory chain, which involve multisubunit complexes of NADH-UQ oxidoreductase, succinate dehydrogenase, UQ-cytochrome c oxidoreductase, cytochrome c oxidase, and ATP synthase. Inhibitor studies show that blocking NADH-UQ oxidoreductase by 1-methyl-4-phenylpyridinium (MPPþ) mimicks Parkinson disease and Leber’s hereditary optic atropy (LHON), whereas inhibition of succinate dehydrogenase by 3-nitropropionic acid (3-NPA) results in the development of symptoms of Huntington’s chorea. LHON is actually caused by single base pair substitution [G/A (Arg!His]) in one of the five NADH dehydrogenase genes. A human X-chromosomal gene also plays a role in determining susceptibility to this mitochondrial defect. Remarkably, expression of the retinal pigment epithelium photoreceptor by an adeno-associated virus vector in LHON models of mice and dogs restored the visual function, providing an example for successful gene therapy of LHON (Acland et al., 2001). Another frequent disease, the Pearson marrow-pancreas syndrome, is caused by mtDNA deletions affecting subunit 4 of NADH dehydrogenase, subunit 1 of cytochrome oxidase and subunit 1 of ATPase, whereas Oncocytoma (responsible primarily for benign solid kidney tumors, which are loaded densely with mitochondria) is caused by deletions in the subunit 1 gene of cytochrome oxidase (Lestienne, 1999; Singh, 1998). Interestingly, mitochondrial diseases causing ATP deficiency can be alleviated by introduction of functional mitochondrial genes into the nucleus (Manfredi et al., 2002). As mentioned in an earlier section, some disease syndromes are linked to mutations affecting mitochondrial translation and DNA replication and repair. For example, mutations in mitochondrial tRNA genes result in mitochondrial myopathy, cardiomyopathy, encephalopathy (MTTL2), myoclonic epilepsy associated with ragged-red fibers (MERRF), and ophthalmoplegia (PEO)—that is, pleiotropic defects (Lestienne, 1999; Singh, 1998). The Kearns-Sayre syndrome/progressive external ophthalmoplegia (KSS/PEO; characterized by pigmentary degeneration of the retina and inflammation of the heart muscles [i.e., cardiomyopathy]) is caused by deletion or base substitution in a gene showing homology to a phage T7 gene-4-like pimase/DNA helicase (Spelbrink et al., 2001) or by deletions affecting DNA polymerase gene (Van Goethem et al., 2001). Interestingly, mitochondrial diseases are also important tools of anthropology and forensic genetics (Budowle et al., 2003), although mtDNA shows limited diversity of genes, but it is rather resistant to decay. The analysis of mtDNA samples recovered from older corpses, ancient

78

Re´dei et al.

relics and bones thus provides valuable information for tracing populations during demographical changes or throughout their evolution (Tishkoff and Williams, 2002).

XI. EPIGENETICS Caspar Wolff (1759) used first the term “epigenesis” as an alternative to preformation. Boveri (1903), who played a major role in experimentally proving the role of chromosomes in heredity, wrote: “alle essentiellen Markmale . . . epigenetisch sind, und daß die Determinierung ihrer Specialita¨t durch den Kern erhalten” (i.e., “all traits are epigenetic and the determination of their characteristics is derived from the nucleus”). During the 1940s Waddington defined the term epigenetics as the study of alteration in the expression of genes during development, in absence of any change affecting the genes themselves. He assumed that meiosis erases epigenetic changes (Waddington, 1953). Epigenetics represented for quite a while a collecting terminology for many genetic observations, which indicated non-Mendelian segregation or unpredictable instability of an apparently mutant allele.

A. Paramutation Paramutation represents one of the classic epigenetic phenomena. A typical paramutation phenomenon is that in certain hybrids carrying a combination of two different, specific alleles, one allele appears as if it can be contaminated by the other (i.e., causes a phenotype, which may or may not be entirely similar to that of the other allele, but seemingly dependent on the other allele). The target allele, which is prone to this effect, is historically named paramutable, and upon undergoing the epigenetic change becomes paramutant (or paramutated). The allele, which causes the epigenetic change, is referred to as the paramutagenic allele. In each epigenetic example, the paramutable and paramutagenic functions proved to be allele-specific; thus no general rule to predict the characteristics of a general paramutagenic allele could be clearly defined by appreciation of classic genetics. In addition, some reports showed that paramutation may also take place at low frequency in the absence of the paramutagenic allele, which further complicated the perception of these events. In addition, it was observed that the penetrance (i.e., expected versus observed segregation ratio) of paramutated alleles widely varied suggesting that the paramutated trait may easily revert during meiosis in some cases (Beck and Olek, 2003; Chandler et al., 2002). One of the classic examples of paramutation was described by Brink (1956) for the maize R locus. He combined the R-r allele, conferring dark purple seed color, with the R-stippled allele, conferring purple stippled seed phenotype.

2. Changing Images of the Gene

79

A proportion of F1 hybrids displayed slight red color and, upon self-fertilization, the reduced coloration trait cosegregated with an apparently modified, paramutated form of the R-r allele, which was designated R-r0 . Thus, it appeared that Rstippled modified in trans the R-r allele. Because the modified R-r0 allele showed varying penetrance, the modification did not appear to be completely stable, suggesting that R-stippled may somehow affect the expression of R-r allele. In addition to the case of R, paramutability of other transcription factor genes (e.g., b1, p1, and pl1) controlling the biosynthesis of flavonoid pigments in seeds and plants were extensively studied in maize. A compilation of paramutation cases in plants is given by Chandler and Stam (2004). Paramutation-like phenomena have been observed also in animals (Herman et al., 2003).

B. Imprinting Imprinting also represents a deviation from Mendel’s rule stating the uniformity and reciprocity of the F1 (i.e., crosses between mutant female with wild type male, and vice versa, result in uniform wild type F1 in case of equal maternal and paternal contribution). Among the many exceptions to this rule, one of the most common is represented by imprinting, which means that the paternal (in most cases) and maternal contributions are unequal. In other words, the paternal and maternal genomes may have different effects (imprinting) on the developing offspring, by modification of expression of one of the alleles, depending on the direction of the cross (in absence of differences among cytoplasmic genes). Thus, in some instances only the female- or only the male-derived genes are imprinted (Bartolomei and Tilghman, 1997), whereas in others, such as that involving the human gene GNAS1 (chromosome 20q13.2-q13.3), biallelic inheritance as well as imprinting is observed in both paternal and maternal directions, depending on the promoter used and alternative splicing (Hayward et al., 1998). Therefore, depending on the effect of one allele on the other, imprinting may be considered a special case of epigenesis (Verona et al., 2003). Imprinting is widespread among species from yeast to humans (de la Casa-Espero´n and Sapienza, 2003). Several examples of imprinting, controlling endosperm development are also known in plants (Choi et al., 2002; Kinoshita et al., 2004). Imprinting is relatively rare; in humans only about 100 or fewer imprinted loci have been identified (Wilkins and Haig, 2003a). These few imprinted loci are known to control essential behavioral, developmental, and sex-specific traits, as well as several important cancer syndromes (Butler et al., 2004; Wilkins and Haig, 2003b). In addition, some nonfamilial (sporadic) human diseases are now also being explained by epigenetic mechanisms (Petronis, 2001). During past decade, many studies focused on imprinting of several human diseases. One of these examples is represented by the Prader-Willi and Angelman syndromes, which are caused by mutations in human chromosome

80

Re´dei et al.

15. The Prader-Willi syndrome generally involves obesity, short stature, poor muscle tension, hypogonadism, and mental retardation, plus compulsive behavior after teenage years. Individuals afflicted by the Angelman syndrome (happy puppet syndrome) usually have protruding tongue and excessive laughter without a cause, defects in motor function, mental retardation, speech defects, epileptic seizures, and so on (Hall, 1997). The locus controlling the disease is in the region 15q11–q13 of the paternal chromosome in the Prader-Willi syndrome, and in the same region of the maternally transmitted chromosome in the Angelman syndrome. Cytological studies show that over 60% of patients in both syndromes are characterized by a chromosome breakage, which marks the so-called imprinting center (IC; Amos-Landgraf et al., 1999). The IC carries sequence duplications permitting unequal crossing over, which leads to deletions of critical regions at 15q11-q13 chromosomal segment and results in methylation, then subsequent silencing of paternally expressed genes in the Prader-Willi syndrome. In some cases, there is no detectable deletion but only a mutation (Hitchins et al., 2004). Imprinting involving sequences within the 15q11-q13 region leads to methylation of the promoter and the first exon of the SNPRN (small ribonucleoprotein) gene, located 35 kb upstream of IC (Perk et al., 2002), as well as histone H3 lysine 4 methylation at the neighboring NDN locus, encoding a protein involved in neuronal differentiation (Lau et al., 2004). In addition, the first exon of a zinc-finger protein gene, ZNF127, located in a more distant region upstream of IC, is methylated at over 100 sites. The ZNF127 sequence is transcribed into an antisense transcript, which is involved in the regulation of other genes in the same region, for example, UBE3 (Jong et al., 1999; Runte et al., 2001). For the Angelman syndrome, analogous DNA rearrangements in the 15q11–q13 region (Wang et al., 2004) lead to a mutation of a gene encoding an E3 ubiquitin ligase E6-associated protein, E6AP (Hitchins et al., 2004). This mutation destroys the function of UBE3/E3A ubiquitin ligase by reducing thiolester formation and substrate ubiquitination. Some mutations, even when retain thiolester formation, are defective in ubiquitination (Cooper et al., 2004). In another widely studied disease of human imprinting is the BeckwithWiedemann syndrome, which causes prenatal overgrowth, macroglossia (enlargement of the tongue), exomphalos (abdominal hernia), and embryonal tumors. This disease is controlled by the insulin-like growth factor II (IGF2) locus in the region 11p15.5, which has two imprinted domains and controlled by two separate imprinting centers. About half of the affected patients express IGF2 from both paternal and maternal alleles; the other 50% display methylation imprinting of a neighboring gene, KCNQ10T1, which codes for a voltage-gated potassium ion channel (Weksberg et al., 2003).

2. Changing Images of the Gene

81

The female transmitted Igf2 allele is imprinted in mouse (The symbol is IGF in humans but Igf in mice). Its imprinting is controlled by the H19 gene, which is located in a distance of 2 Mb from Igf2 and IGF2, respectively. H19 is also a target for imprinting, but its alleles show different levels of methylation in the two sexes (Bartolomei and Tilghman, 1997). Imprinting of H19 is of regulatory relevance, because H19 is transcribed to a noncoding RNA, which negatively controls transcription of its targets. During development the Igf2 gene is expressed from three different promoters in the paternal chromosome, and thus its imprinting changes developmentally according to promoter choice (Bartolomei and Tilghman, 1997; Vu and Hoffman, 1994). In the choroid plexus (brain tissue secreting the cerebrospinal fluid) and the leptomeninges (the innermost of the three membranes covering the brain and the spinal chord) Igf2 is not imprinted (DeChiara et al., 1991). Similarly, during tumorigenesis both paternal and maternal copies of the Igf2 gene are expressed, but at different stages; the paternal copy at early, the maternal copy at later stages (Vernucci et al., 2004). Igf2 shows preferential expression in the male because a germlineinherited methylation silences the promoter of H19 gene. The 50 upstream region of H19 contains an imprinting methylation signal (called mark) in the male, a 42-bp element, which appears to be conserved in mammals (Jones et al., 2001; Sparago et al., 2004). IGF2/Igf2 and homologs are imprinted in humans, rodents, sheep, and pig; but it is biallelically expressed in chickens (Haig, 2004). The regulatory mechanism of IGF2 shows some analogy to inactivation of the multiple X-chromosomal genes, in mouse and humans. In early embryonic tissues, most paternally inherited X-chromosomal genes are inactive, except the Xist gene of mouse (the human homolog is TSIX), which is expressed only from the paternally derived X chromosome (Duthie et al., 1999). This gene has functional similarity to H19, inasmuch as it is not translated; but its RNA transcript inactivates the X chromosomal genes present in more than one copy (Wutz et al., 2002).

C. Mechanisms of epigenetic modifications As illustrated by examples in an earlier section, epigenetic modification of gene expression involves several mechanisms. Molecular analysis of many imprinted genes indicate that DNA methylation, and methylation of lysine residues in histone tails play major roles in gene silencing by imprinting (de la CasaEspero´n and Sapienza, 2003; Kaneda et al., 2004; Verona et al., 2003). Numerous examples from plants and animals indicate that methylation-induced gene silencing can result in a lasting condition, epimutation (i.e., the methylation is not erased in meiosis, but it is detectable in the embryos following fertilization). As discussed in an earlier section, methylation of the target genes may be

82

Re´dei et al.

transmitted by either maternal or paternal parents, or by both (Frevel et al., 1999; Rakyan et al., 2003). However, meiotic transmission of the epigenetic state was also found to occur in fission yeast and Drosophila, organisms that show no or low levels of DNA methylation, respectively. Thus, clearly other mechanisms must also operate in stable transmission of epimutations. As illustrated by the cited examples, as well as by other genetic studies of fungi and plants (Chandler and Stam, 2004)—in addition to DNA methylases, noncoding antisense and small RNAs (i.e., involved in repeat-induced gene silencing, which leads to dsRNA formation and silencing through RNAi) are also implicated in stable maintenance of epimutations. Studies in fission yeast and Arabidopsis clearly indicate that repeat-induced RNAi-mediated silencing is functionally interlinked with histone H3 Lys9 methylation, which plays a major role in heterochromatin assembly and epigenetic gene silencing (Grewal and Rice, 2004; Matzke et al., 2004). An RNAi effector complex RITS (RNA-induced initiator of transcriptional gene silencing) has been identified in fission yeast, which targets siRNAs to specific DNA sequences promoting heterochomatin assembly (Verdel et al., 2004). This complex contains Ago1 (a member of Argonaute family proteins, which bind siRNAs), a chromodomain protein Chp1, and its interacting partner Tas3. This RITS complex is thought to recruit histone H3 Lys29 methylase (and possibly other histone methylases) in a chromatin context, and to interact with heterochromatin-binding Polycomb and chromodomain factors, such as SWI6/HP1 (Brehm et al., 2004; Verdel et al., 2004; Vermaak et al., 2003). Alteration of the organization of chromatin structure, gene silencing and activation, including inhibition of enzymes, manipulating methyltransferases and histone methylation, and use of siRNA, are thus all within the realm of epigenetics (Egger et al., 2004). Another crucial question to answer is how epigenetic modifications pass through DNA replications. Imprinted genes usually replicate asynchronously from the remainder of the genome. Although it appears that expressed genes replicate early, this rule does not seem to hold for imprinting because early replicating paternal genes may still be silent. For example, in mice the Mest, Peg3, Snrpn, Ndn, Impact, Ig2r, and Kip genes are involved in imprinting, and these are turned on and off separately during oogenesis (Obata and Kono, 2002). Some understanding of mechanisms involved in meiotic transmission of epimutations was derived from observations that DNA methylation and histone H3 K9-specific methylation are functionally interlinked. This has been demonstrated by genetic studies revealing that ubiquitous DNA methylation is lost in Neurospora and in mouse embryonic stem cell mutants defective in histone H3-K9 methylases (Lehnertz et al., 2003; Tamaru and Selker, 2001). Moreover, in other systems it was shown that histone H3-K9 methylation is dependent on DNA methylation (Fahrner et al., 2002; Tariq et al., 2003),

2. Changing Images of the Gene

83

indicating that these two major forms of epigenetic modifications are interdependent. A flurry of papers show that DNA methyltransferases interact with proliferating cell nuclear antigen (PCNA), a component of DNA replication complex, and that C-methyl–binding proteins can recruit histone H3-K9 methylases to the chromatin assembly factor CAF-1. It is thus likely that coordinated maintenance of DNA and histone H3-K9 methylation of imprinted genes at heterochromatic loci are controlled during DNA replication by DNA and histone methylases and methyl-CpG–binding factors, which constitute components of potentially interacting DNA replication–chromatin assembly complexes (Sarraf and Stancheva, 2004).

XII. PRIONS Prions (protein infectious agents) represent a unique class of biologically active molecules that are apparent exceptions to the nucleic-based replication/multiplication mechanisms (Prusiner, 2004). Prions representing the causative agents of transmissible spongiform encephalopathies (TSE) are infective and transmitted from cell to cell within an organism (Chien et al., 2004; Weissmann, 1999). In humans a prion-like protein (PrPC) is coded by two exons of the PRNP gene in the 20pter-p12 chromosomal region, but analogous PrPC coding genes have also been described in other vertebrates, as well as in fungi (Prusiner, 2004). Hundreds of PRNP gene mutations have been identified, some of which were shown to result in the generation of protease resistant infective prions, termed PrPSc (Fig. 2.2; Weissmann, 1999). The Sc superscript was named after the scrapie encephalitis of sheep; the disease causes the animals to scrape their bodies against sharp objects for invisible causes of extreme itching. Certain mutations in the prion gene or genes (e.g., sheep have two prion genes) make more likely the development of one or the other type of infective diseases. Prions are self-propagating proteins. The likely mechanism is that infectious PrPSc protein binds to and converts its normal cellular counterpart PrPC to an abnormal form by inducing conformational changes and subsequent polymerization into amyloid fibers. The protein tangle is deposited in larger quantities mainly in the outermost membranes of the brain and results in destruction of glial and other brain cells (Scott et al., 1999). There is a fundamental difference between genes and prions. When the prion protein PrPSc infects and converts the normal protein of another organism (e.g., infection of cattle by sheep prion), the primary structure of protein of the recipient remains largely unaltered but some -helices are converted into sheets; and only the conformation of the donor is imposed on the host protein

84

Re´dei et al.

(Prusiner, 2004). Several cellular factors, such as molecular chaperons, facilitate self-propagation of prions through this “protein-only” multiplication reaction. How prions incite frightening TSE syndromes, such as scrapie, bovine spongiform encephalopathy (BSE), or the Creutzfeldt–Jakob disease, is not clear but seem to be correlated with posttranslational modification and potential membrane targeting of PrPC. The basic prion protein has two N-linked glycosylation sites and a glycosylphosphatidyl inositol anchor (GPI). Both the normal PrPC and the prion PrPSc contain at least 52 N-linked oligosaccharides, although in different relative proportions because the abnormal acetylglucoseaminyl transferase III protein machinery is perturbed (Rudd et al., 1999). The GPI anchor in prions is modified with sialic acid. The latter compound appears to secure mobility in lipid bilayers, and it is present in ganglions of the brain. Thus, the anchor can provide means for translocation of the proteins from cell to cell— comprising infectivity, a characteristic feature of prion diseases (Rudd et al., 2001). The low efficiency of PrPSc-induced amyloid polymerization reactions requires homotypic aggregation and propagation a so-called “prion-forming” domain (PrD), and various elements of which have been characterized in several fungal prion proteins (Tutie and Koloteva-Levin, 2004). Multiplication of PrPSc by imparting its conformation onto the noninfective PrPC protein predicted that rodent mutants carrying an inactive Prnp gene would be resistant to prion diseases. In fact, Prnp0/0 knockout mice turned out to be fully resistant to prion infection, providing a proof for the aforementioned protein-only hypothesis of Prusiner (Bu¨eler et al., 1993). Additional evidence further supports this protein-only principle. A synthetic peptide (free of nucleic acids), containing mutation at site 102 leucine (Fig. 2.2) folded into a -conformation-rich form, was introduced into mice; such animals developed disease homologous to the Gerstmann–Sta¨ussler syndrome in humans (Tremblay et al., 2004). Furthermore, the peptide-induced disease was serially passaged into healthy mice, which developed symptoms indistinguishable from those appearing spontaneously in PrPSc leucine mutants. Similarly, recombinant protein consisting of the mouse prion sequence 89–231, rich in -sheets, was cloned in E. coli and then introduced into the brain of animals that overexpressed the normal PrPC. Mice developed neuropathological symptoms characteristic of specific encephalopathy, and their protease-resistant extract evoked disease symptoms in other animals. There were two essential differences from the normal infection. The responding recipients produced excessive amounts of PrPC before inoculation, and the period of incubation was substantially extended compared with the normal course of disease development (Legname et al., 2004). Although nucleic acids-free infectious agents evoke disease symptoms, mammalian RNAs, but not invertebrate RNAs, may be adjuvants in

2. Changing Images of the Gene

85

Figure 2.2. A segment of the human prion gene, within which mutations resulting in amino acid replacements may favor development of one of the best-known human prion diseases. (Octa means 8-nucleotide sequence, e.g., ATTTGCAT that frequently occurs in the promoters of other eukaryotic genes too.)

development of disease peptides (Deleault et al., 2003). The latest evidence for protein being the causative agent is rather convincing (see in an earlier section). However, some scientists seem to be reluctant to accept the protein-only idea without reservations (Couzin, 2004). Behavior of prions can be studied with greater ease in fungi, in which several different types of prions have been identified. In fungi, the prion systems are somewhat different from those in mammals. Yet, two of the five human PrP repeats (PHGGWGQ) can substitute for the yeast PSIþ repeats in stabilizing the yeast prion aggregate, indicating the evolutionary conservation of the N-termini (Parham et al., 2001). Nuclear genes of URE2 and SUP35 are responsible for the cytoplasmic prion proteins, URE3 and PSIþ. The C-terminal domain of the URE gene

86

Re´dei et al.

product is involved in the metabolism of ureidosuccinate, and the N-terminal region is concerned with prion conformation. The SUP35 gene encodes a subunit of the eukaryotic translation termination complex (eRF3). When the N-terminal region of the Urep protein was lost, prion activity was lost too; and when the amino end of the regulatory Urep protein was overexpressed, prion production increased thousands-fold. PSI is the product of the SUP35 gene, and it controls the aggregation of the prion subunits into active form. New evidence shows that the amino-terminal fragment of PSIþ alone can be sufficient to bring about anomalies of prion conformation (Tanaka et al., 2004).

XIII. CONCLUSIONS AND OUTLOOK Our views about the gene have undergone dynamic and dramatic changes. Although nucleic acids were discovered in the nineteenth century, since 1953 their meaning has revolutionized biology. The past half-century witnessed outstanding discoveries and linked technical developments, including the definitive identification of the genetic material, discovery of the mechanism of coding, establishment of gene synthesis methods, recombinant DNA technologies, complete nucleotide sequencing of an impressive set of both small and large genomes. The polymerase chain reaction now permits rapid amplification of genes or parts of genes (e.g., Innis et al., 1999), and the expression of thousands of genes can be simultaneously and routinely analyzed from small samples by nucleic acid hybridization on microarrays (Ermolaeva et al., 1998). Genetic information transfer no longer relies on sexual means only—genes can be otherwise transferred among many different types of organisms, ranging from viruses to higher eukaryotes. The combination of formerly separate disciplines permits large-scale analyses of gene-networks and interactions of encoded protein modules. The spectacular success of the gene theory was accomplished by the fortunate application of inductive and deductive logic, including the combination of empirical methods of classic biology with mathematics and biochemistry. Despite the pessimistic—or perhaps overly optimistic—views expressed by some biologists that most of the major features of genes and genetics have already been discovered, the literature suggests that genetics is reincarnated, by combining the “old” information and consolidating it with bioinformatics and proteomics (Re´dei, 2003). Genetics and gene theory, by continuously finding new foci of research, such as systems biology, development, olfactogenetics, neurobiology, evolution, and its ever widening applications in medicine, agriculture, forensics, and so on, remain as vigorous and exciting as ever.

2. Changing Images of the Gene

87

XIV. EPILOG Personal comments and anecdotes also illustrate the changing images of the gene concept. Edwin Cohn, the physical chemist asked T. H. Morgan in the late 1920s about his research plans. Morgan, the first Nobel laureate in genetics, answered that “I am not doing any genetics, I am bored with genetics. But I am going out to Cal Tech where I hope it will be possible to bring physics and chemistry to bear on biology.” Shortly after Morgan arrived to Cal Tech, Albert Einstein visited the laboratory and posed about the same question. Morgan’s answer was about the same as before. Einstein shook his head and said: “No, this trick won’t work. The same trick does not work twice. How on earth are you ever going to explain in terms of chemistry and physics so important a biological phenomenon as first love?” (Borsook, 1956). Morgan’s views in the 1920s foreshadowed the coming trend of combining the study of genes with biochemistry. The humorous response of Einstein suggests that neither he nor Morgan could anticipate—as one of the many possible examples—what was to emerge from modern neurogenetics, one element of which involves impressive correlations of “behavioral genes” with functions of nerve synapses and with a host of other molecular-neurobiological phenomena (Hall, 2003). Francis Crick (1978) recalled other revealing anecdotes: “Paul Doty told me that shortly after lapel buttons came in he was in New York and to his astonishment saw one with “DNA” written on it. Thinking it must refer to something else he asked the vendor what it meant. “Get with it, bud” the man replied in a strong New York accent, “dat’s the gene.” Crick also remembered “An even odder incident happened when Jim [Watson] came back to work at Cambridge in 1955. I was going into the Cavendish one day and found myself walking with Neville Mott, the new Cavendish professor [Bragg had gone on to the Royal Institution in London]). “I’d like to introduce you to Watson” I said, “since he’s working in your lab.” He looked at me in surprise. “Watson?” he said. “Watson? I thought your name was Watson-Crick.” To characterize the state of the gene concept, we cite the Nobel laureate Walter Gilbert (2003): “It seems to me that molecular biology is dead. DNA-based thinking has penetrated the whole of biology, and the separate field no longer exists. Also gone is the attempt to answer broad fundamental questions—how does DNA work? What controls a gene?—by single individuals. However, in fractal fashion, new sciences appear in the details as we continue to learn. Science is both an individual and a collective endeavor. Like the artist, the creative individual finds new discoveries—most often manifest at the moments of breakthrough, when an idea reveals a new field of knowledge—that characterize its forefront.”

88

Re´dei et al.

Acknowledgments We thank George P. Smith, James A. Birchler, David Sleper, and Magdi Re´dei for comments and suggestions.

REFERENCES Acland, G. M., Aguirre, G. D., Ray, J., Zhang, Q., Aleman, T. S., Cideciyan, A. V., Pearce-Kelling, S. E., Anand,V., Zeng,Y., Maguire, A. M., Jacobson, S. G., Hauswirth, W. W., et al. (2001). Gene therapy restores vision in a canine model of childhood blindness. Nat. Genet. 28, 92–95. Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F., George, R. E., Lewis, S. E., et al. (2000). The genome sequence of Drosophila melanogaster. Science 287, 2185–2195. Agol, I. J. (1929). Stepallelomorphism in Drosophila melanogaster. Zhurnal Eksperimentalnoi Meditsiny 5, 86–101. Albert, R., Jeong, H., and Baraba´si, A.-L. (2000). Error and attack tolerance of complex networks. Nature 406, 378–382. Amos-Landgraf, J. M., Ji, Y., Gottlieb, W., Depinet, T., Wandstrat, A. E., Cassidy, S. B., Driscoll, D. J., Rogan, P. K., Schwartz, S., and Nicholls, R. D. (1999). Chromosome breakage in the Prader-Willi and Angelman syndromes involves recombination between large, transcribed repeats at proximal and distal breakpoints. Am. J. Hum. Genet. 65, 370–386. Avery, O. T., MacLeod, C. M., and McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal type III. J. Exp. Med. 79, 137–158. Balakirev, E. S., and Ayala, J. F. (2003). Pseudogenes: Are they “junk” or functional DNA? Annu. Rev. Genet. 37, 123–151. Baltimore, D. (1970). RNA-dependent DNA polymerase in virions of R tumour viruses. Nature 226, 1209. Baraba´si, A.-L., and Albert, R. (1999). Emergence of scaling in random networks. Science 286, 509–512. Baraba´si, A. L., and Oltvai, Z. N. (2004). Network biology: Understanding the cell’s functional organization. Nat. Rev. Genet. 5, 101–113. Bar-Joseph, Z., Gerber, G. K., Lee, T. I., Rinaldi, N. J., Yoo, J. Y., Robert, F., Gordon, D. B., Fraenkel, E., Jaakkola, T. S., Young, R. A., and David K Gifford, D. K. (2003). Computational discovery of gene modules and regulatory networks. Nat. Biotechnol. 21, 1337–1342. Barrell, B. G., Bankier, A. T., and Druin, J. A. (1979). A different genetic code in the human mitochondria. Nature 282, 189–194. Bartel, D. P. (2004). MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 116, 281–297. Bartolomei, M. S., and Tilghman, S. M. (1997). Genomic imprinting in mammals. Annu. Rev. Genet. 31, 493–525. Bateson, W. (1902). “Mendel’s Principle of Heredity: A Defence.” University Press, Cambridge, UK. Bateson, W. (1926). Segregation: Being the Joseph Leidy memorial lecture at the university of Pennsylvania. J. Genet. 16, 201–235. Bateson, W., and Saunders, E. R. (1902). Experimental studies in the physiology of heredity. Rep. Evol. Comm. R. Soc. 1, 1–160. Battersby, B. J., Loredo-Ost, J. C., and Shoubridge, E. A. (2003). Nuclear genetic control of mitochondrial DNA segregation. Nat. Genet. 33, 183–186.

2. Changing Images of the Gene

89

Baur, E. (1909). Das Wesen und die Erblichtskeitverha¨ltnisse der “varietates albomarginatae hort.” von Pelargonium zonale. Zeitschr. Ind. Abstamm Vererb-Lehre 1, 330–351. Beadle, G. W. (1945). Biochemical genetics. Chem. Rev. 37, 15–96. Beadle, G. W. (1957). The role of nucleus in heredity. In “A Symposium on the Chemical Basis of Heredity” (W. D. McElroy and B. Glass, eds.), pp. 3–22. Johns Hopkins Press, Baltimore, MD. Beck, S., and Olek, A. (eds.) (2003). “The Epigenome. Molecular Hide and Seek.” Wiley-VCH, Wienheim, Germany. Belling, J. (1928). The ultimate chromomeres of Lilium and Aloe with regard to the numbers of genes. Univ. California Publ. Bot. 14, 307–318. Benzer, S. (1957). The elementary units of heredity. In “A Symposium on the Chemical Basis of Heredity” (W. D. McElroy and B. Glass, eds.), pp. 70–93. Johns Hopkins University Press, Baltimore, MD. Berg, D. E., and Howe, M. M. (eds.) (1989). “Mobile DNA.” American Society of Microbiology, Washington, DC. Berget, S. M., Berk, A. J., Harrison, T., and Sharp, P. A. (1977). Spliced segments at the 50 termini of adenovirus-2 late mRNA: A role for heterogeneous nuclear RNA in mammalian cells. Cold Spring Harb. Symp. Quant. Biol. 42, 523–529. Berka, R. M., Cui, X., and Yanofsky, C. (2003). Genome-wide transcriptional changes associated with genetic alterations and nutritional supplementation affecting tryptophan metabolism in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 100, 5682–5687. Bessho, Y., Hodgson, D. R., and Suga, H. A. (2002). tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme. Nat. Biotechnol. 20, 723–728. Birky, C. W., Jr. (2001). The inheritance of genes in mitochondria and chloroplasts: Laws, mechanisms, and models. Annu. Rev. Genet. 35, 125–148. Borsook, H. (1956). Informal remarks “by way of summary.” J. Cell. Comp. Physiol. 47(Suppl. 1), 283–286. Boucher, Y., Douady, C. J., Papke, R. T., Walsh, D. A., Boudreau, M. E. R., Nesbø, C. L., Case, R. J., and Ford Doolittle, W. (2003). Lateral gene transfer and the origins of prokaryotic groups. Annu. Rev. Genet. 37, 283–328. ¨ ber den Einfluß der Samenzelle auf die Larvencharactere der Echiniden. Roux’ Boveri, T. (1903). U Arch. Entwicklungsmech. Organism 16, 340–363. Brehm, A., Tufteland, K. R., Aasland, R., and Becker, P. B. (2004). The many colours of chromodomains. Bioessays. 26, 133–140. Brenner, S. (1993). Thoughts on genetics at the fin de sie`cle. Trends Genet. 9, 104. Brent, R., Russel, R., and Finley, L., Jr. (1997). Understanding gene and allele function with twohybrid methods. Annu. Rev. Genet. 31, 663–704. Bridges, C. B. (1923). The translocation of chromosome II upon chromosome III. in Drosophila. Anat. Rec. 24, 426–427. Bridges, C. B. (1936). The Bar “gene” a duplication. Science 83, 210–211. Brink, R. A. (1956). A genetic change associated with the R locus in maize which is directed and potentially reversible. Genetics 41, 872–889. Brockmann, G. A., Kratzsch, J., Haley, C. D. S., Renne, U., Schwerin, M., and Karle, S. (2000). Single QTL effects, epistasis, and pleiotropy account for two-thirds of the phenotypic F92 variance of growth and obesity in DU6i x DBA/2 mice. Genome Res. 10, 1941–1957. Broker, T. R., Chow, L. T., Dunn, A. R., Gelinas, R. E., Hassel, J. A., Klessig, D. F., Lewis, J. B., Roberts, R. J., and Zain, B. S. (1977). Adenovirus-2 messengers: An example of baroque molecular architecture. Cold Spring Harb. Symp. Quant. Biol. 42, 531–553. Budowle, B., Allard, M. W., Wilson, M. R., and Chakraborty, R. (2003). Forensics and mitochondrial DNA: Applications, debates, and foundations. Annu. Rev. Genomics Hum. Genet. 4, 119–141.

90

Re´dei et al.

Bu¨eler, H., Aguzzi, A., Sailer. A., Greiner, R. A., Autenried, P., Aguet, M., and Weissmann, C. (1993). Mice devoid of PrP are resistant to scrapie. Cell 73, 1339–1347. Butler, J. A. V. (1952). The nucleic acids of the chromosomes. Endeavour 11, 154–158. Butler, M. G., Bittel, D. C., Kibiryeva, N., Talebizadeh, Z., and Thompson, T. (2004). Behavioral differences among subjects with Prader-Willi syndrome deletion and maternal disomy. Pediatrics 113, 565–573. Capecchi, M. R. (1980). High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22, 479–488. Carlson, E. A. (1966). “The Gene: A Critical History.” W. B. Saunders, Philadephia, PA. Carlson, E. A. (1981). “Genes, Radiation, and Society. The Life and Work of H. J. Muller.” Cornell University Press, Ithaca, NY. Carlson, M., and Botstein, D. (1982). Two differentially regulated mRNAs with different 50 ends encode secreted and intracellular forms of yeast invertase. Cell 28, 145–154. Carvalho, A. B. (2002). Origin and evolution of the Drosophila Y chromosome. Curr. Opin. Genet. Dev. 12, 664–668. Catcheside, D. G. (1960). Relation of genotype to enzyme content. In “Microbial Genetics” (W. Hayes and R. C. Clowes, eds.), pp. 181–207. Cambridge University Press, Cambridge, UK. Cawley, S., Nekiranov, S., Ng, H. H., Kapranov, P., Sekinger, E. A., Kampa, D., Piccolboni, A., Sementchenko, V., Cheng, J., Williams, A. J., Wheeler, R., Wong, B., et al. (2004). Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116, 499–509. Chandler, V. L., Stam, M., and Sidorenko, L. V. (2002). Long-distance cis and trans interactions mediate paramutation. Adv. Genet. 46, 215–234. Chandler, V. L., and Stam, M. (2004). Chromatin conversations: Mechanisms and implications of paramutation. Nat. Rev. Genet. 5, 532–545. Chargaff, E. (1955). On the chemistry and function of nucleoproteins and nucleic acids. Istituto Lombardo (Rend. Sc.) 89, 101–115. Chargaff, E. (1979). How genetics got a chemical education. Ann. N. Y. Acad. Sci. 325, 345–360. Chargaff, E., Vischer, E., Doniger, R., Grenn, C., and Misani, F. (1949). The composition of the deoxypentose nucleic acids of thymus and spleen. J. Biol. Chem. 177, 405–416. Chen, K., Gray, J. C., and Wildman, S. G. (1975). Fraction 1 protein and the origin of polyploid wheats. Science 190, 1304–1305. Chien, P., Weissman, J. S., and DePace, A. H. (2004). Emerging principles of conformation-based prion inheritance. Annu. Rev. Biochem. 73, 617–656. Choi, T., Huang, M., Gorman, C., and Jaenisch, R. (1991). A generic intron increases gene expression in transgenic mice. Mol. Cell. Biol. 11, 3070–3074. Choi, Y., Gehring, M., Johnson, L., Hannon, M., Harada, J., Goldberg, R. B., Jacobsen, S. E., and Fischer, R. L. (2002). DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110, 33–42. Cohen, S. N. (1976). Transposable genetic elements and plasmid evolution. Nature 263, 731–738. Cohen, S. N., Chang, A. C. Y., and Hsu, L. (1972). Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69, 2110–2114. Coin, L., Bateman, A., and Durbin, R. (2003). Enhanced protein domain discovery by using language modeling techniques from speech recognition. Proc. Natl. Acad. Sci. USA 100, 4516–4520. Collins, F. S., Green, E. D., Guttmacher, A. E., and Guyer, M. S. (2003). A vision of the future of genomics research. Nature 422, 835–847.

2. Changing Images of the Gene

91

Cooper, E. M., Hudson, A. W., Amos, J., Wagstaff, J., and Howley, P. M. (2004). Biochemical analysis of Angelman syndrome-associated mutations in the E3 ubiquitin ligase E6-associated protein (E6AP). J. Biol. Chem. 279, 41208–41217. Correns, C. (1904). Experimentelle Untersuchungen u¨ber Gynodioecy. Ber. Dtsch. Bot. Ges. 22, 506–517. Correns, C. (1909). Zur Kenntnis der Rolle von Kern und Plasma bei der Verbung. Zeitschr. Ind. Abstamm. Vererb.-Lehre 2, 331–340. Couzin, J. (2004). An end to the prion debate? Don’t count on it. Science 305, 589. Craig, N. L. M., Craigie, R., Gellert, M., and Lambowitz, A. (eds.) (2002). “Mobile DNA II.” Am. Soc. Microbiol. Press, Washington, DC. Crick, F. H. C. (1958). On protein synthesis. Symp. Soc. Exp. Biol. 12, 138–163. Crick, F. (1978). How to live with a golden helix. The Sciences 19(7), 6–9. Date, A. V., and Marcotte, E. M. (2003). Discovery of uncharacterized cellular systems by genomewide analysis of functional linkages. Nat. Biotechnol. 21, 1055–1062. DeChiara, T. M., Robertsoon, E. J., and Efstratiades, E. (1991). Parental imprinting of the mouse insulinlike growth factor II gene. Cell 64, 849–859. De Greve, H., Lemans, J., Harnalsteens, J. P., Thia-Toong, L., De Beuckeleer, M., Willmitzer, L., Olten, L., Van Montagu, M., and Schell, J. (1982). Regeneration of normal and fertile plants that express octopine synthase, from tobacco crown galls after deletion of tumour-controlling functions. Nature 300, 752–755. de la Casa-Espero´n, E., and Sapienza, C. (2003). Natural selection and the evolution of genome imprinting. Annu. Rev. Genet. 37, 349–370. Deleault, N. R., Lucassen, R. W., and Supattpone, S. (2003). RNA molecules stimulate prion protein conversion. Nature 425, 717–720. Demerec, M. (1955). What is a gene? Twenty years later. Am. Nat. 89, 5–20. Demerec, M. (1967). Properties of genes. In “Heritage from Mendel” (R. A. Brink, ed.), pp. 49–61. University of Wisconsin Press, Madison, WI. Demerec, M., and Demerec, Z. E. (1956). Analysis of linkage relationships in Salmonella by transduction techniques. Brookhaven Sym. Biol. 8, 75–87. Dillon, N. (2003). Positions, please . . .. Nature 425, 457. Dubinin, N. P. (1929). Allelomorphentreppen bei Drosophila melanogaster. Biol. Zbl. 49, 328–339. Dubinin, N. P., and Sidorov, B. N. (1934). Relation between the effect of a gene and its position in the system. Am. Nat. 68, 377–381. Dubinin, N. P., and Sidorov, B. N. (1935). The position effect of the hairy gene. Biol. Zh. 4, 455–468. Duthie, S. M., Nesterova, T. B., Formstone, E. J., Keohane, A. M., Turner, B. M., Zakian, S. M., and Brockdorff, N. (1999). Xist RNA exhibits a banded localization on the inactive X chromosome and is excluded from autosomal material in cis. Hum. Mol. Genet. 8, 195–204. Egger, G., Liang, G., Aparicio, A., and Jones, P. A. (2004). Epigenetics in human disease and prospects for epigenetic therapy. Nature 429, 457–463. Elseth, G. G., and Baumgardner, K. D. (1984). “Genetics.” Addison-Wesley, Reading, MA. Ermolaeva, O., Rastogi, M., Pruitt, K. D., Schuler, G. D., Bittner, M. L., Chen, Y., Simon, R., Meltzer, P., Jeffrey, M., Trent, J. M., and Boguski, M. S. (1998). Data management and analysis for gene expression arrays. Nat. Genet. 20, 19–23. Fahrner, J. A., Eguchi, S., Herman, J. G., and Baylin, S. B. (2002). Dependence of histone modifications and gene expression on DANN hypermethylation in cancer. Cancer Res. 62, 7213–7218. Feng, Q., Zhang, Y., Hao, P., Wang, S., Fu, G., Huang, Y., Li, Y., Zhu, J., Liu, Y., Hu, X., Jia, P., Zhang, Y., et al. (2002). Sequence and analysis of rice chromosome 4. Nature 420, 316–320.

92

Re´dei et al.

Ferrall, M. (2004). Quantitative genetic variation: A post-modern view. Hum. Mol. Genet. 13, Spec. No. 1:R1–R7. Fields, S., and Song, O. K. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245–246. Fire, A. (1999). RNA-triggered gene silencing. Trends Genet. 15, 358–363. Fraenkel-Conrat, H., and Williams, R. C. (1955). Reconstitution of active tobacco mosaic virus from its inactive protein and nucleic acid components. Proc. Natl. Acad. Sci. USA 41, 690–698. Frevel, M. A. E., Sowerby, S. J., Petersen, G. B., and Reeve, A. E. (1999). Methylation sequencing analysis refines the region of H19 epimutation in Wilms tumor. J. Biol. Chem. 274, 29331–29340. Garcia-Bellido, A., and Ripoll, P. (1978). The number of genes in Drosophila melanogaster. Nature 273, 399–400. Gavin, A.-C., Bo¨sche, M., Krause, R., Grandi. P., Marzioch, M., Bauer, A., Schultz, J., Rick, J. M., Michon, A.-M., Cruciat, C.-M., Remor, M., Ho¨fert, C., et al. (2002). Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147. Gerull, B., Gramlich, M., Atherto, J., McNabb, M., Trombitas, K., Sasse-Klaassen, S., Seidman, J. G., Seidman, C., Granzier, H., Labeit, S., Frenneaux, M., and Thierfelder, L. (2002). Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy. Nat. Genet. 30, 201–204. Gierer, A., and Mundry, K. W. (1958). Production of mutants of tobacco mosaic virus by chemical alteration of its ribonucleic acid in vitro. Nature 182, 1457–1458. Gilbert, W. (1978). Why genes in pieces? Nature 271, 501. Gilbert, W. (2003). Life after the helix. Nature 421, 315–316. Giles, N. H. (1951). Studies on the mechanism of reversion in biochemical mutants of Neurospora. Cold Spring Harb. Symp. Quant. Biol. 16, 283–313. Giot, L., Bader, S. J., Brouwer, C., Chaudhuri, A., Kuang, B., et al. (2003). A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736. Goldschmidt, R. (1958). “Theoretical Genetics.” University of California Press, Berkeley, CA. Gonza´lez-Pastor, J. E., San Milla´n, J. L., and Moreno, F. (1994). The smallest known gene. Nature 369, 281. Gordon, J. W., and Ruddle, F. H. (1981). Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214, 1244–1246. Gowen, J. W., and Gay, E. H. (1933). Gene number, kind and size in Drosophila. Genetics 18, 1–31. Grewal, S. I. S., and Rice, J. C. (2004). Regulation of heterochromatin by histone methylation of small RNAs. Curr. Opin Cell Biol. 16, 230–238. Griffith, F. (1928). Significance of pneumococcal types. J. Hyg. 27, 113–159. Guo, J., Casolaro, V., Seto, E., Yang, W.-M., Chang, C., Seminario, M.-C., Keen, J., and Georas, S. N. (2001). Ying-Yang activates interleukin-4 gene expression in T cells. J. Biol. Chem. 276, 48871–48878. Hagemann, R. (2002). Milestones in plastid genetics of higher plants. Prog. Bot. 63, 5–51. Haig, D. (2004). Genomic imprinting and kinship: How good is the evidence. Annu. Rev. Genet. 38, 553–585. Hall, J. C. (2003). A neurogeneticist’s manifesto. J. Neurogenet. 17, 1–90. Hall, J. G. (1997). Genomic imprinting: Nature and clinical relevance. Annu. Rev. Med. 48, 35–44. Hammarsten, O. (1894). Zur Kenntnis der Nukleoproteide. Hoppe Seyler’s Zeitschr. Physiol. Chem. 19, 19–37. Hanson, F. B. (1928). The effect of X-rays in producing return gene mutation. Science 67, 562–563. Hao, B., Gong, W., Ferguson, T. K., James, C. M., Krzycki, J. A., and Chan, M. K. (2002). A new UAG-encoded residue in the structure of a methanogen methyltransferase. Science 29, 1462–1466.

2. Changing Images of the Gene

93

Harpending, H., and Rogers, A. (2000). Genetic perspectives on human origins and differentiation. Annu. Rev. Genomics Hum. Genet. 1, 361–385. Hartwell, L. H., Hopfield, J. J., Leibler, S., and Murray, A. W. (1999). From molecular to modular cell biology. Nature 402(Suppl.), C47–C52. Hayward, B. E., Moran, V., Strain, L., and Bonthron, D. T. (1998). Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins. Proc. Natl. Acad. Sci. USA 95, 15475–15480. He, L., and Hannon, G. J. (2004). MicroRNAs: Small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531. Herman, H., Lu, M., Anggraini, M., Sikora, A., Chang, Y., Yoon, B. J., and Soloway, P. D. (2003). Trans allele methylation and paramutation-like effects in mice. Nat. Genet. 34, 199–202. Herrera-Estrella, L., Depicker, A., Van Montagu, M., and Schell, J. (1983). Expression of chimeric genes transferred into plant cells using Ti plasmid-derived vector. Nature 303, 209–213. Hershey, A. D., and Chase, M. (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36, 39–56. Hertwig, O. (1875). Beitra¨ge zur Kenntnis der Bildung, Befruchtung und Theilung der tierischen Eies. Morphologisches Jahrbuch 1, 347–437. Hertwig, O. (1895). “The Cell. Outlines of General Anatomy and Physiology.” Macmillan, New York. Hirotsune, S., Yoshida, N., Chen, A., Garrett. L., Sugiyama, F., Takahashi, S., Yagami, K., Wynshaw-Bori, A., and Yoshiki, A. (2003). An expressed pseudogene regulates the messengerRNA stability of its homologous coding gene. Nature 423, 91–96. Hitchins, M. P., Dhalla, R. S., de Vries, B. B., Winter, R., Pembrey, M. E., and Malcolm, S. (2004). Investigation of UBE3A and MECP2 in Angelman syndrome (AS) and patients with features of AS. Am. J. Med. Genet. 125A, 167–172. Hogness, D. S., Lipshitz, H. D., Beachy, P. A., Peattie, D. A., Saint, R. B., Goldschmidt-Clermont, M., Harte, P. J., Gavis, E. R., and Helfand, S. L. (1985). Regulation and products of the Ubx domain of the bithorax complex. Cold Spring Harb. Symp. Quant. Biol. 50, 181–194. Hollis, G. F., Hiete, P. A., McBride, O. W., Swan, D., and Leder, P. (1982). Processed genes: A dispersed human immunoglobulin gene bearing evidence of RNA-type processing. Nature 296, 321–325. Honjo, T., Nakai, S., Nishida, Y., Kataoka, T., Yamawaki-Kataoka, Y., Takahashi, N., Obata, M., Shimizu, A., Yaoita, Y., Nikaido, T., and Ishida, N. (1981). Rearrangements of immunoglobulin genes during differentiation and evolution. Immunol. Rev. 59, 33–67. Hood, L., and Galas, D. (2003). The digital code of DNA. Nature 421, 444–448. Hood, L., Heath, J. R., Phelps, M. E., and Lin, B. (2004). Systems biology and new technologies enable predictive and preventive medicine. Science 306, 640–643. Hotchkiss, R. D. (1957). The role of desoxyribonucleates in bacterial transformation. In “Phosphorus Metabolism” (W. D. MacElroy and B. Glass, eds.), Vol. 2, pp. 426–436. Johns Hopkins Press, Baltimore, MD. Hutva´gner, G., and Zamore, P. D. (2002). A microRNA in a multiple-turnover RNAi enzyme complex. Science 297, 2056–2060. Ideker, T., Ozier, O., Schwikowski, B., and Siegel, A. F. (2002). Discovering regulatory and signalling circuits in molecular interaction networks. Bioinformatics (Suppl. 1), S233–S240. Innis, M., Gelfand, D., and Sninsky, J. (eds.) (1999). “PCR Applications: Protocols for Functional Genomics.” Academic Press, San Diego, CA. International Human Genome Sequencing Consortium (2004). Finishing the euchromatic sequence of the human genome. Nature 431, 931–945. Jackson, A. L., and Linsley, P. S. (2004). Noise amidst the silence: Off-target effects of siRNAs? Trends Genet. 20, 521–524.

94

Re´dei et al.

Jackson, D., Symon, R. H., and Berg, P. (1972). Biochemical method for inserting new genetic information into DNA of Simian virus 40: Circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 69, 2904–2909. Jacob, F., and Monod, J. (1961). Genetic regulatory mechanism in the synthesis of proteins. J. Mol. Biol. 3, 318–356. Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987). GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907. Johannsen, W. (1909). “Elemente der exakten Erblichkeitslehre.” Jena, Fischer, Germany. Jones, B. K., Levorse, J., and Tilghman, S. M. (2001). Deletion of a nuclease-sensitive region between Igf2 and H19 genes leads to Igf2 misregulation and increased adiposity. Hum. Mol. Genet. 10, 807–814. Jong, M. T. C., Gray, T. A., Ji, Y., Glenn, C. C., Saitoh, S., Driscoll, D. J., and Nicholls, R. D. (1999). A novel imprinted gene, encoding a RING zinc-finger protein, and overlapping antisense transcript in the Prader-Willi syndrome critical region. Hum. Mol. Genet. 8, 783–793. Jordan, E., Saedler, H., and Starlinger, P. (1968). O-zero and strong polar mutations in the gal operon are insertions. Mol. Gen. Genet. 10, 353–363. Judson, H. F. (1979). “The Eighth Day of Creation.” The Makers of the Revolution in Biology. Simon and Schuster, New York. Kamalay, J. C., and Goldberg, R. B. (1980). Regulation of structural gene expression in tobacco. Cell 19, 935–946. Kamp, R. M., Calvete, J. J., and Choli-Papadopoulou, T. (eds.) (2004). “Methods in Proteome and Protein Analysis.” Springer-Verlag, Berlin. Kaneda, M., Okano, M., Hata, K., Sado, T., Tsujimoto, N., Li, E., and Sasak, H. (2004). Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429, 900–903. Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., and Kakutani, T. (2004). One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation. Science 303, 521–523. Klein, T. M., Wolf, E. D., Wu, R., and Sanford, J. C. (1987). High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327, 70–93. Klug, W. S., and Cummings, M. R. (1983). “Concepts of Genetics.” Merrill, Columbus, OH. Knight, R. D., Freeland, S. J., and Landweber, L. F. (2001). Rewiring the keyboard: Evolvability of the code. Nat. Rev. Genet. 2, 49–58. Koncz, C., Olsson, O., Langridge, W. H. R., Schell, J., and Szalay, A. A. (1987). Expression and assembly of functional bacterial luciferase in plants. Proc. Natl. Acad. Sci. USA 84, 131–135. Korotkov, K. V., Novoselov, S. V., Hatfield, D. L., and Gladyshev, N. V. (2002). Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec insertion sequence element. Mol. Cell. Biol. 22, 1402–1411. Lau, J. C., Hanel, M. L., and Wevrick, R. (2004). Tissue-specific and imprinted modifications of the human NDN gene. Nucleic Acids Res. 32, 3376–3382. Lazowska, J., Jaq, C., and Slonimski, P. P. (1980). Sequence of introns and flanking exons in wildtype and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron. Cell 22, 333–348. Lederberg, J. (1959). In “Genetics, Genetic Information and the Control of Protein Structure and Function” (H. E. Sutton, ed.), p. 164. Joshiah Macey Jr. Foundation, Madison, NJ. Legname, G., Baskakov, I. V., Nguyen, H.-O. B., Riesner, D., Cohen, F. E., DeArmond, S. J., and Prusiner, S. B. (2004). Synthetic mammalian prions. Science 305, 673–676. Lehnertz, B., Ueda, Y., Derijck, A. A., Braunschweig, U., Perez-Burgos, L., Kubicek, S., Chen, T., Li, E., Jenuwein, T., and Peters, A. H. (2003). Suv39h-mediated histone H3 lysine 9 methylation

2. Changing Images of the Gene

95

directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr. Biol. 13, 1192–1200. Leister, D., and Schneider, A. (2003). From genes to photosynthesis in Arabidopsis thaliana. Int. Rev. Cytol. 228, 31–83. Lestienne, P. (1999). “Mitochondrial Diseases.” Springer, New York. Levene, P. A., and Lawrence, W. B. (1931). “Nucleic Acids.” The Chemical Catalog Company Inc, New York. Lewin, B. (2004). “Genes VIII.” Prentice Hall, New York. Lewis, E. B. (1945). The relation of repeats to position effect in Drosophila melanogaster. Genetics 30, 137–166. Locke, J., and Tartof, K. D. (1994). Molecular analysis of cubitus interruptus (ci) mutations suggests an explanation for unusual position effects. Mol. Gen. Genet. 243, 234–243. Manfredi, G., Fu, J., Ojaimi, J., Sadlock, J. E., Kwong, J. Q., Guy, J., and Schon, E. A. (2002). Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat. Genet. 30, 394–395. Marcotte, E. M., Pellegrini, M., Ng, H.-L., Rice, D. W., Yeats, T. O., and Eisenberg, D. (1999). Detecting protein function and protein-protein interactions from genome sequences. Science 285, 751–753. Marshall, R. E., Caskey, T. C., and Nirenberg, M. (1967). Fine structure of the RNA codewords recognized by bacterial, amphibian, and mammalian transfer RNA. Science 155, 820–826. Martens, J. A., Laprade, L., and Winston, F. (2004). Intergenic transcription is required to repress the Saccharomyces cerevisiae SER3 gene. Nature 429, 571–574. Maslov, S., and Sneppen, K. (2002). Specificity and stability in topology of protein networks. Science 296, 910–913. Matzke, M., Aufsatz, W., Kanno, T., Daxinger, L., Papp, I., Mette, M. F., and Matzke, A. J. (2004). Genetic analysis of RNA-mediated transcriptional gene silencing. Biochim. Biophys. Acta 1677, 129–141. McAlister-Henn, L., Gibson, N., and Panisko, E. (1999). Applications of the yeast two-hybrid system. Methods 19, 330–337. McClintock, B. (1942). The fusion of broken ends of chromosomes following nuclear fusion. Proc. Natl. Acad. Sci. USA 28, 458–463. McClintock, B. (1956). Controlling elements and the gene. Cold Spring Harb. Symp. Quant. Biol. 21, 197–216. Meller, V. H., and Kuroda, M. I. (2002). Sex and the single chromosome. Adv. Genet. 46, 1–24. ¨ ber die chemische Zusammensetzung der Eiterzellen. Hoppe Seyler’s Med. Miescher, F. (1871). U Chem. Untersuch. 4, 441–460. Miescher, F. (1874). Die Spermatozoen einiger Wirbelthiere. Ein Beitrag zur Histochemie. Verhandl. Naturforsch. Ges. Basel. 6(I), 138–208. Migula, W. (1897). System der Bakterien. Handbuch der Morphologie, Entwicklungsgeschichte und Systematik der Bakterien. Jena, Fischer. Miller, W. J., and Capy, P. (eds.) (2004). “Mobile Genetic Elements.” Humana, Totowa, NJ. Mirsky, A. E., and Pollister, A. W. (1946). Chromosomin, a deoxyribose nucleoprotein complex of the cell nucleus. J. Gen. Physiol. 10, 117–147. Modrek, B., and Lee, C. J. (2003). Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat. Genet. 34, 177–180. Moraes, C. T. (1998). Characteristics of mitochondrial DNA diseases. In “Mitochondrial DNA Mutations in Aging, Disease and Cancer” (K. K. Singh, ed.), pp. 167–184. Springer, Berlin. Morgan, T. H. (1926). Genetics and the physiology of development. Am. Nat. 60, 489–515. Morgan, T. H., Sturtevant, A. H., Muller, H. J., and Bridges, C. B. (1915). “The Mechanism of Mendelian Heredity.” Holt & Co., New York.

96

Re´dei et al.

Muller, H. J. (1929). The gene as the basis of life. Proc. Int. Cong. Plant Sci. 1, 897–921. Muller, H. J. (1950). The development of the gene concept. In “Genetics in the 20th Century” (L. C. Dunn, ed.), pp. 77–99. Macmillan, New York. Na¨geli, C. W. (1867). Cited by Mendel in a letter to Na¨geli on 18 April 1867. Genetics 33(Suppl.), 5. Na¨geli, C. W. (1884). “Mechanisch-physiologische Theorie der Abstammungslehre.” Mu¨nchen, Germany. Newton, K. J., Mariano, J. M., Gibson, C. M., Kuzmin, E., and Gabay-Laughnan, S. (1996). Involvement of S2 episomal sequences in the generation of NCS4 deletion mutation in maize mitochondria. Dev. Genet. 19, 277–286. Nirenberg, M. W., and Matthaei, H. (1961). The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polynucleotides. Proc. Natl. Acad. Sci. USA 47, 1588–1602. Nott, A., Meislin, S. H., and Moore, M. J. (2003). A quantitative analysis of intron effects on mammalian gene expression. RNA 9, 607–617. Obata, Y., and Kono, T. (2002). Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth. J. Biol. Chem. 277, 5285–5289. Ochoa, S., Burma, D. P., Kroger, H., and Weill, J. D. (1961). Proc. Natl. Acad. Sci. USA. 47, 670–679. Offermann, C. A. (1935). The position effect and its bearing on genetics. Izv. Akad. Nauk. USSR Ser 7, 129–140. Olby, R., and Posner, E. (1967). An early reference to genetic coding. Nature 215, 556. Oliver, C. P. (1940). A reversion to wild-type associated with crossing-over in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 26, 452–454. Orgel, L. E., and Crick, F. H. (1980). Selfish DNA: The ultimate parasite. Nature 284, 604–607. Orgel, L. E., Crick, F. H., and Sapienza, C. (1980). Selfish DNA. Nature 288, 645–646. Paddison, P. J., Silva, J. M., Conclin, D. S., Schlabach, M., Li, M., Areuleba, S., Balija, V., O’Shaughnessy, A., Gnoj, L., Scobie, K., Chang, K., Westbook, T., et al. (2004). A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431. Parham, S. N., Resende, C. G., and Tuite, M. F. (2001). Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions. EMBO J. 20, 2111–2119. Park, K.-S., Lee, D.-K., Lee, H., Lee, Y., Kim, Y. H., Yang, H.-Y., Lee, S.-I., Seol, W., and Kim, J.-S. (2003). Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors. Nat. Biotechnol. 21, 1208–1214. Patel, A. A., and Steitz, J. A. (2003). Splicing double: Insights from the second spliceosome. Nat. Rev. Mol. Cell Biol. 4, 960–970. Patthy, L. (1999). Genome evolution and the evolution of exon shuffling: A review. Gene 238, 103–114. Pauling, L., and Corey, R. B. (1953). A proposed structure for the nucleic acids. Proc. Natl. Acad. Sci. USA 39, 84–97. Pennisi, E. (2004). Searching for the genome’s second code. Science 306, 632–635. Periasamy, M., Strehler, E. E., Garfinkel, L. I., Gabits, R. M., Ruiz-Opazo, N., and Nadal-Ginard, B. (1984). Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing. J. Biol. Chem. 259, 13595–13604. Perk, J., Makedonski, K., Lande, L., Cedar, H., Razin, A., and Shemer, R. (2002). The imprinting mechanism of the Prader-Willi/Angelman regional control center. EMBO J. 21, 5807–5814. Petronis, A. (2001). Human morbid genetics revisited: Relevance of epigenetics. Trends Genet. 17, 142–146. Pierce, B. (2003). “Genetics. A Conceptual Approach.” Freeman, New York. Plate, L. (1913). “Vererbungslehre.” Engelmann, Leipzig, Germany.

2. Changing Images of the Gene

97

Preer, J. R., Jr. (1963). Discussion. In “Methodology of Basic Genetics” (W. J. Burdette, ed.), p. 374. Holden-Day, San Francisco. Prusiner, S. B. (ed.) (2004). “Prion Biology and Diseases.” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Rakyan, V. K., Chong, S., Champ, M. E., Cuthbert, P. C., Morgan, H. D., Luu, K. V. K., and Whitelaw, E. (2003). Transgenerational inheritance of epigenetic states at the murine AxinFu allele occurs after maternal and paternal transmission. Proc. Natl. Acad. Sci. USA 100, 2538–2543. Ravasz, E., Somera, A. L., Mongru, D. A., Oltvai, Z. N., and Baraba´si, A. L. (2002). Hierarchical organization of modularity in metabolic networks. Science 297, 1551–1555. Re´dei, G. P. (1971). A portrait of John Lewis Stadler. Stadler Genet. Symp. 1, 1–20. Re´dei, G. P. (2003). “Encyclopedic Dictionary of Genetics, Genomics, and Proteomics.” Wiley-Liss, Hoboken, NJ. Re´dei, G. P., Acedo, G., and Sandhu, S. S. (1984). Mutation induction and detection. In “Mutation, Cancer, and Malformation” (E. H. Y. Chua and W. M. Generoso, eds.), pp. 285–313. Plenum, New York. Rudd, P. M., Endo, T., Colominas, C., Groth, D., Wheeler, S. F., and Harvey, D. J. (1999). Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc. Natl. Acad. Sci. USA 96, 13044–13049. Rudd, P. M., Wormald, M. R., Wing, D. R., Prusiner, S. B., and Dwek, R. A. (2001). Prion glycoprotein: Structure, dynamics, and roles for sugars. Biochemistry 40, 3759–3766. Runte, M., Hu¨ttenhofer, A., Groß, S., Kiefmann, M., Horsthemke, B., and Buiting, K. (2001). The IC-SNURF–SNRPN transcript serves as a host for multiple small nucleolar RNA species and as an antisense RNA for UBE3A. Hum. Mol. Genet. 10, 2687–2700. Sakharkar, M. K., Chow, V. T., and Kangueane, P. (2004). Distributions of exons and introns in the human genome. Silico Biol. 4(2), 0032 (electronic preprint, June 16, 2004). Sarraf, S. A., and Stancheva, I. (2004). Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol. Cell 15, 595–605. Sasaki, T., Matsumoto, T., Yamamoto, K., Sakata, K., Baba, T., Katayose, Y., Wu, J., Niimura, Y., Cheng, Z., Nagamura, Y., Antonio, B. A., Kanamori, H., et al. (2002). The genome sequence and structure of rice chromosome 1. Nature 420, 312–316. Schibler, U., and Sierra, F. (1987). Alternative promoters in developmental gene expression. Annu. Rev. Genet. 21, 237–257. Schneider, M., Ow, D. W., and Howell, S. H. (2000). The in vivo pattern of firefly luciferase expression in transgenic plants. Plant Mol. Biol. 14, 935–947. Schro¨dinger, E. (1967). “What is Life? The Physical Aspect of the Living Cell.” Cambridge University Press, London. Scott, M. R., Will, R., Ironside, J., Nguyen, H.-O. B., Tremblay, P., DeArmond, S. J., and Prusiner, S. B. (1999). Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proc. Natl. Acad. Sci. USA 96, 15137–15142. Seitz, H., Youngson, N., Lin, S.-P., Dalbert, S., Paulsen, M., Bachellerie, J.-P., Ferguson-Smith, A. C., and Cavaille´, J. (2003). Imprinted microRNA genes transcribed antisense to a reciprocally imprinted retrotransposon-like gene. Nat. Genet. 34, 261–262. Singer, M., and Berg, P. (1991). “Genes & Genomes. A Changing Perspective.” University Science Books, Mill Valley, CA. Singh, K. K. (ed.) (1998). “Mitochondrial DNA, Mutations in Aging, Disease and Cancer.” Springer, New York. Snyder, M., and Gerstein, M. (2003). Genomics. Defining genes in the genomics era. Science 300, 258–260.

98

Re´dei et al.

Solzhenitsyn, A. I. (1973). “The Gulag Archipelago.” Harper & Row, New York. Sparago, A., Cerrato Vernucci, M., Ferrero, G. B., Silengo, M. C., and Riccio, A. (2004). Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome. Nat. Genet. 36, 958–960. Spelbrink, N., Li, F.-Y., Tiranti, V., Nikali, K., Yuan, Q.-P., Tariq, M., Wanrooij, S., Garrido, N., Comi, G., Morandi, L., Santoro, L., Toscano, et al. (2001). Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat. Genet. 28, 223–231. Stadler, L. J. (1946). Spontaneous mutation at the R locus in maize I. The aleuron-color and plantcolor effects. Genetics 31, 377–391. Stadler, L. J. (1954). The gene. Science 120, 811–819. Stadler, L. J., and Roman, H. (1948). The effect of X-rays upon mutation of the gene A in maize. Genetics 33, 273–303. Stadler, L. J., and Uber, F. M. (1942). Genetic effects of ultraviolet radiation in maize. IV. Comparison of monochromatic radiations. Genetics 27, 84–118. Stent, G. S. (1969). “The Coming of the Golden Age. A View of the End of Progress.” Natural History Press, Garden City, NY. Stern, C. (1973). “Principles of Human Genetics.” Freeman, San Francisco, CA. Stolc, V., Gauhar, Z., Mason, C., Halasz, G., van Batenburg, M. F., Rifkin, S. A., Hua, S., Herremen, T., Tongprasit, W., Barbano, P. R., Bussemaker, H. J., and White, K. P. (2004). A gene expression map for he euchromatic genome of Drosophila melanogaster. Science 306, 655–660. ¨ ber Befruchtung und Zelltheilung.” Jena, Germany, Dabis. Strasburger, E. (1875). “U Stupar, R. M., Lilly, J. W., Town, C. D., Cheng, Z., Kaul, S., Buell, C. R., and Jiang, J. (2001). mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: Implication of potential sequencing errors caused by large-unit repeats. Proc. Natl. Acad. Sci. USA 98, 5099–5103. Sturtevant, A. H. (1913). The linear arrangement of sex-linked factors in Drosophila, as shown by their association. J. Exp. Zool. 14, 43–59. Sturtevant, A. H. (1925). The effects of unequal crossing over at the bar locus in Drosophila. Genetics 10, 117–147. Sturtevant, A. H. (1926). A crossover reducer in Drosophila melanogaster due to inversion of a section of the third chromosome. Biol. Zbl. 46, 697–702. Sturtevant, A. H. (1965). “A History of Genetics.” Harper & Row, New York. Sturtevant, A. H., and Beadle, B. W. (1939). “An Introduction to Genetics.” Constable & Co, London, UK. Surpin, M., Larkin, R. M., and Chory, J. (2002). Signal transduction between the chloroplast and the nucleus. Plant Cell 14(Suppl.), S327–S338. Tanaka, M., Chien, P., Naber, N., Cooke, R., and Weissman, J. S. (2004). Conformational variations in an infectious protein determine prion strain differences. Nature 428, 323–328. Tamaru, H., and Selker, E. U. (2001). A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414, 277–283. Tariq, M., Saze, H., Probst, A. V., Lichota, J., Habu, Y., and Paszkowszli, J. (2003). Erasure of CpG methylation in Arabidopsis alters patterns of histone methylation of heterochromatin. Proc. Natl. Acad. Sci. USA 100, 8823–8827. Temin, H. M. (1964). Homology between RNA from Rous sarcoma virus and DNA from Rous sarcoma-infected cells. Proc. Natl. Acad. Sci. USA 52, 323–329. Tennyson, C. N., Klamut, H. J., and Worton, R. G. (1995). The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nat. Genet. 9, 184–190. Tenson, T., Xiong, L., Kloss, P., and Mankin, A. S. (1997). Erythromycin resistance peptides selected from random peptide libraries. J. Biol. Chem. 272, 17425–17430.

2. Changing Images of the Gene

99

Thorsness, P. E., and Weber, E. R. (1996). Escape and migration of nucleic acids between chloroplasts, mitochondria, and the nucleus. Int. Rev. Cytol. 165, 207–234. Timofe´eff-Ressovsky, N. W. (1930). Das Genovariieren in verschiedenen Richtungen bei Drosophila melanogaster unter dem Einfluß der Ro¨ntgenbestrahlung. Naturwissenschaften 18, 434–437. ¨ ber die Natur der GenmuTimofe´eff-Ressovsky, N. V., Zimmer, K. G., and Delbru¨ck, M. (1935). U tation und Genstruktur. Nachricht. Ges. Wissenschaft. Go¨ttingen, Math. Phys. Kl., Biol. 1, 189–245. Tishkoff, S. A., and Williams, S. M. (2002). Genetic analysis of African populations: Human evolution and complex disease. Nat. Rev. Genet. 3, 611–621. Tremblay, P., Ball, H. L., Kaneko, K., Groth, D., Hegde, R. S., Cohen, F. E., DeArmond, S. J., Prusiner, S. B., and Safar, J. G. (2004). Mutant PrPSc conformers induced by a synthetic peptide and several prion strains. J. Virol. 78, 2088–2099. Troland, L. T. (1917). Biological enigmas and the theory of enzyme action. Am. Nat. 51, 321–350. Tsien, R. Y. (1998). The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544. Tutie, M. F., and Koloteva-Levin, N. (2004). Propagating prions in fungi and mammals. Cell 14, 541–552. Van Beneden, E. (1883). Recherches sur la maturation de l’oeuf et la fe´condation. Arch. Biol. 4, 265–640. Van Goethem, G., Dermaut, B., Lo¨fgren, A., Martin, J.-J., and Van Broeckhoven, C. (2001). Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions. Nat. Genet. 28, 211–212. Varona, L., Gomez-Raya, L., Rauw, W. M., Clop, A., Ovilo, C., and Noguera, J. L. (2004). Genetics 166, 1025–1035. Venter, J. C., Adams, M. D., Myers, E. W., Li, P., Mural, W. R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A., Gocayne, J. D., Amanatides, P., et al. (2001). The sequence of the human genome. Science 291, 1304–1351. Verdel, A., Jia, S., Gerber, S., Sugiyama, T., Gygi, S., Grewal, S. I., and Moazed, D. (2004). RNAimediated targeting of heterochromatin by the RITS complex. Science 303, 672–676. Vermaak, D., Ahmad, K., and Henikoff, S. (2003). Maintenance of chromatin states: An open-andshut case. Curr. Opin. Cell Biol. 15, 266–274. Vernucci, M., Cerrato, F., Pedone, P. V., Dandolo, L., Bruni, C. B., and Riccio, A. (2004). Developmentally regulated functions of the H19 differentially methylated domain. Hum. Mol. Genet. 13, 353–361. Verona, R. I., Mann, M. R. W., and Bartolomei, M. S. (2003). Genomic imprinting: Intricacies of epigenetic regulation in clusters. Annu. Rev. Cell Dev. Biol. 19, 237–259. Vu, T. H., and Hoffman, A. R. (1994). Promoter-specific imprinting of the human insulin-like growth factor-II gene. Nature 372, 464–467. Waddington, C. H. (1953). Epigenetics and evolution. Symp. Soc. Exp. Biol. 7, 186–199. Walker, E. L., Robbins, T. P., Bureau, T. E., Kermicle, J., and Dellaporta, S. L. (1995). Transposonmediated chromosomal rearrangements and gene duplications in the formation of the maize R-r complex. EMBO J. 14, 2350–2363. Wang, J., Hamilton, J. I., Carter, M. S., Li, S., and Wilkinson, M. F. (2002). Alternatively spliced TCR mRNA induced by disruption of reading frame. Science 297, 108–110. Wang, N. J., Liu, D., Parokonny, A. S., and Schannen, N. C. (2004). High- resolution characterization of 15q11-q13 rearrangements by array comparative hybridization (array CGH) with detection of gene dosage. Am. J. Hum. Genet. 75, 267–281. Watson, J. D., and Crick, F. H. C. (1953a). Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 171, 737–738. Watson, J. D., and Crick, F. H. C. (1953b). Genetical implications of the structure of deoxyribonucleic acid. Nature 171, 964–967.

100

Re´dei et al.

Weissmann, C. (1999). Molecular genetics of transmissible spongiform encephalopathies. J. Biol. Chem. 274, 3–6. Weksberg, R., Smith, A. C., Squire, J., and Sadowski, P. (2003). Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Hum. Mol. Genet. 12, R61–R68. Weston, A. D., and Hood, L. J. (2004). Systems biology, proteomics, and the future of health care: Toward predictive, preventative, and personalized medicine. Proteome Res. 3, 179–196. Wilkins, J. F., and Haig, D. (2003a). What good is genomic imprinting: The function of the parentspecific gene expression. Nat. Rev. Genet. 4, 359–369. Wilkins, J. F., and Haig, D. (2003b). Inbreeding, maternal care and genomic imprinting. J. Theor. Biol. 221, 559–564. Williams, M. E., and Levings, C. S., III (1992). Molecular biology of cytoplasmic male sterility. Plant Breeding 10, 23–51. ¨ . (1958). Wilhelm Johannsen. J. Hered. 4, 83–88. Winge, O Witkin, E. M. (2002). Chances and choices: Cold spring harbor 1944–1955. Annu. Rev. Microbiol. 56, 1–15. Wolff, C. F. (1759). “Theoria Generationis.” Halle, Germany. Woltereck, R. (1909). Weitere experimentelle Untersuchungen u¨ber Artvera¨nderung speciell u¨ber das Wesen quantitativer Unterschiede der Daphniden. Dtsch. Zool. Ges. 110–172. Wutz, A., Rasmussen, T. P., and Jaenisch, R. (2002). Chromosomal silencing and localization are mediated by different domains of Xist RNA. Nat. Genet. 30, 167–174. Xia, Y., Yu, H., Jansen, R., Seringhausk, M., Baxter, S., Greenbaum, D., and Zhu, H. (2004). Analyzing cellular biochemistry in terms of molecular networks. Annu. Rev. Biochem. 73, 1051–1087. Xu, Q., Modrek, B., and Lee, C. (2002). Genome-wide detection of tissue-specific alternative splicing in the human transcriptome. Nucleic Acids Res. 30, 3754–3766. Yamada, K., Lim, J., Dale, J. M., Chen, H., Shinn, P., Palm, P. J., Southwick, A. M., C. Wu, H. C., Kim, C., Nguyen, M., Pham, P., Cheuk, R., et al. (2003). Empirical analysis of transcriptional activity of the Arabidopsis genome. Science 302, 842–846. Yandell, M. D., and Majoros, W. H. (2002). Genomics and natural language processing. Nat. Rev. Genet. 3, 601–610. Yanofsky, C. (1963). Amino acid replacements associated with mutation, recombination in the gene A and their relationship to in vitro coding data. Cold Spring Harb. Symp. Quant. Biol. 28, 581–588. Yc˘as, M. (1969). “The Biological Code.” Wiley & Sons, New York. Zheng, L., Liu, J., Batalov, S., Zhou, D., Orth, A., Ding, S., and Schultz, P. D. (2004). An approach to genome-wide screens of expressed small interfering RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 135–140. Zhu, H., Bilgin, M., and Snyder, M. (2003). Proteomics. Annu. Rev. Biochem. 72, 783–812.

3

Historical and Modern Genetics of Plant Graft Hybridization Yongsheng Liu Department of Horticulture Henan Institute of Science and Technology Xinxiang 453003, China

I. Introduction II. Historical Background A. Darwin: The first to put forward the conception of graft hybridization B. Winkler, Daniel, and Burbank’s studies of graft hybridization C. Michurin’s contribution to graft hybridization III. The Existence of Graft Hybrids A. Further evidence for graft hybrids B. Reasons why graft hybrids have been met with skepticism IV. Methods of Graft Hybridization A. Production of chimera graft hybrids B. Mentor-grafting procedures V. Characteristics of Graft Hybridization A. Systems based on chimera graft hybrids B. Systems based on nonchimera graft hybrids VI. Mechanisms Underlying Graft Hybridization A. Darwin’s Pangenesis, the first scientific explanation B. Different viewpoints on the formation of graft hybrids C. A new perspective on the mechanism of graft hybridization VII. Significance of Graft Hybridization A. The phenomena considered with regard to plant genetics in general B. The phenomenon considered from the perspective of plant breeding Advances in Genetics, Vol. 56 Copyright 2006, Elsevier Inc. All rights reserved.

0065-2660/06 $35.00 DOI: 10.1016/S0065-2660(06)56003-1

Yongsheng Liu

102 VIII. Conclusions Acknowledgments References

ABSTRACT Graft hybridization is a type of asexual hybridization in which heritable changes may be induced by grafting. Darwin was the first to put forward the conception of graft hybridization. The existence of graft hybrids has been extensively documented, although there has been a refusal to accept its reality, other than perceiving the phenomenon as involving “simple” chimeras. Graft hybrids can be divided into two categories—chimera graft hybrid (so-called graft chimera) and nonchimera graft hybrid (so-called vegetative hybrid). These differ with respect to grafting methods, characteristics, and mechanisms proposed to underlie the two categories. Graft hybridization is not only a simple and powerful means of plant breeding but also provides striking evidence in favor of Darwin’s notions about Pangenesis—a developmental theory of heredity, on the one hand, and a phenomenon that plays a crucial role in revealing the mystery of non-Mendelian inheritance in grafted fruit trees. ß 2006, Elsevier Inc.

I. INTRODUCTION Grafting as a means of vegetative propagation is well known. Less well known is that genes may move between the stock and the “scion” (defined in a later section) by grafting, thus resulting in heritable changes. In 1868, Darwin coined the term graft hybrid and graft hybridization, which he believed to be striking evidence in favor of his theory of heredity—Pangenesis. In the second edition of his Variation of Animals and Plants under Domestication, he wrote: “I will therefore give all the facts which I have been able to collect on the formation of hybrids between distinct species or varieties, without the intervention of the sexual organs. For if, as I am now convinced, this is possible, it is a most important fact, which will sooner or later change the view of physiologists with respect to sexual reproduction” (Darwin, 1883). “Sooner or later” has proved to be a long time, and the fulfillment of Darwin’s conditional prophecy is not yet complete (Michie, 1958). Most biologists have recognized the existence of somatic hybrids produced by

3. Plant Graft Hybridization

103

protoplast fusion since 1970s. However, there has been a refusal to accept the existence of graft hybrids, which have been regarded only as a type of chimera. Do graft hybrids really exist? Does graft hybridization—if it occurs at all—play a significant role in plant genetics and breeding? These questions have been the subject of passionate scientific debate since the late nineteenth century (Crane, 1949; Dean, 1962; Flegr, 2002; Hagemann, 2002; Jouin, 1900; Liu, 2004a,b; Weiss, 1930). The decisive successes of classic genetics and molecular biology submerged this debate (Landman, 1991). The following statements are typical in their dismissal of the true meaning of graft hybrid: “Graft hybrid: a type of plant chimera that may be produced when a part of one plant (the scion) is grafted onto another plant of a different genetic constitution (the stock). Shoots growing from the point of union of the graft contain tissues from both the stock and the scion” (Martin and Hine, 2000). “Nonchimeric type graft hybrids were postulated. . . . called . . . also vegetative hybrids, and claimed that grafting alters the hereditary material of both graft and scion. These claims were not reproducible by appropriate methods of experimentation, and several of the results were due either to ignorance or deliberate deception” (Redei, 1998). If asked, most biologists today would say that graft hybridization never occurs. Such situation is not often encountered in natural science—a near-consensus among scientific specialists on a viewpoint that is squarely contradicted by a substantial body of reliable experimental evidence (Landman, 1993). This chapter, however, reconsiders the subject of graft hybridization in light of our present understanding. Thus, observations concerning graft hybridization will be discussed in a context that is compatible with concepts of molecular genetics—that graft hybridization and sexual hybridization can coexist comfortably in the universe of Darwin’s Pangenesis and molecular biology.

II. HISTORICAL BACKGROUND A. Darwin: The first to put forward the conception of graft hybridization The origins of grafting can be traced to ancient times. The practice of grafting was carried on by the Chinese thousands of years before the Christian era (Daniel,

104

Yongsheng Liu

1929; Hartmann et al., 1997; Zhou, 1994). There is also evidence that graftinduced changes were known to the Chinese more than 1400 years ago (Liu, 2000). According to Daniel, the typical recorded case of the occurrence of a graft hybrid is that mentioned in 1578 by Shi-Zhen Li, in his book Ben Cao Gang Mu (Chinese Medicinal Herbs), who described a tree occurring in China formed by grafting a scion of plum onto a stock of peach. This tree produced fruits exhibiting a mixture of the characters of the two components of the graft (Weiss, 1930). Although there were many records of graft-induced changes from time to time in horticultural literature, the conception of graft hybridization and graft hybrids was first put forward by Darwin. In Chapter 11 of his book The Variation of Animals and Plants under Domestication, Darwin gave all the facts he had collected on the formation of graft hybrids, that is, individuals produced from the united cellular tissue of two different plants. He began with the famous case of Adam’s laburnum (Cytisus adami), which was made by grafting a bud of the purple laburnum (Cytisus purpureus) into a stock of the common laburnum (Cytisus laburnum). It bore three kinds of flowers—some dingy-red, some large and bright yellow, others small and purple. That is to say, it bore its own hybrid flowers, also those of its two parents. Then Darwin introduced Bizzarria orange, which was produced from a seedling that had been grafted. This curious plant produced on some of its branches bitter orange, on others citron, and on some fruits that were partly bitter orange and partly citron. He also mentioned grafts to jessamine, oleander, ash, hazel, grape, hyacinths, rose, and especially to potatoes. From a host of facts, Darwin not only believed that under certain unknown conditions graft hybridization could be effected but also was convinced of the reality and importance of graft hybridization. At the end of the section, Darwin wrote: “Finally it must, I think, be admitted that we learn from the foregoing cases a highly important physiological fact, namely, that the elements that go to the production of a new being, are not necessarily formed by the male and female organs. They are present in the cellular tissue in such a state that they can unite without the aid of the sexual organs, and thus give rise to a new bud partaking of the characters of the two parent-forms.” Darwin opposed the idea that all the recorded cases were attributed to simple bud variation because when varieties are produced by simple bud variation, they frequently present quite new characters; whereas the graft hybrids are intermediate in character between the two forms employed. To explain the formation of graft hybrids, the inheritance of acquired characters, xenia (the action of male element on the tissues of mother plant), reversion, and many other facts pertaining to heredity, variation, propagation, regeneration, and

3. Plant Graft Hybridization

105

development, Darwin presented his “provisional hypothesis of Pangenesis.” In addition, it is likely that Darwin had realized that graft hybridization was the probable mechanism for the noninheritance phenomenon in grafted fruit trees. He noticed that “certain fruit trees truly propagate their kind while growing on their own roots; but when grafted on other stocks, and by this process their natural state is manifestly affected, they produce seedlings which vary greatly, departing from the parental type in many characters.” There has been a lack of agreement in the literature over proper terminology for the phenomenon described in this chapter. Darwin (1883) called it “graft hybridization”; Michurin (1949), “graft hybridization” or “vegetative hybridization”; Ohta (1991) and Hirata et al. (2003), “graftoduction” or “graft transformation.” I prefer to use “graft hybridization” in honor of Darwin’s pioneer work in this field.

B. Winkler, Daniel, and Burbank’s studies of graft hybridization Winkler grafted tomato and nightshade in an attempt to generate graft hybrid. From grafts of the two very distinct plants, several types of shoots were regenerated from the callus at the graft junction. Most of the shoots were identical to either tomato or nightshade, but some showed a blending of nightshade and tomato characters, which Winkler first thought were graft hybrids. Winkler also obtained shoots in which the main axis was composed of tomato tissue in a sector adjacent to nightshade tissue. He called this a “graft chimera,” after the “Chimera” of Greek mythology. In a later paper, Winkler arrived at the following conclusion—hybrids may be arranged in two groups, sexual hybrid and graft hybrid. The latter may be divided into three classes according to the theoretical possibility of their method of origin: (1) Fusion graft hybrid arising from a fusion of two somatic cells derived from distinct species. (2) “Influenced” graft hybrid arising from specific influences of one graft component upon the other without cell fusion. (3) Chimera, in which specifically pure cells from both graft components are combined to form a new individual (cited in Bailey, 1920). Daniel devoted more than 36 years to the study of grafting and other horticultural operations. He described many cases in which, as a result of grafting, a shoot was produced combining characters of scion and stock, and held that such combinations of characters in a shoot could be inherited. He also illustrated that, following a grafting operation, new heritable variations could appear, which show features not present in either scion or stock. He possessed the specimens of the perennial varieties that he had obtained and distributed some cuttings or tubers of these to scientists who wished to study them. Daniel believed that his results not only proved the accuracy of Lamarck’s hypothesis pertaining to the inheritance of acquired characters, due to changing environment, but also demonstrated that, in certain cases, grafting is a powerful agent

106

Yongsheng Liu

for causing the formation of new varieties (Daniel, 1929; Neilson-Jones, 1969). It is worth mentioning that, in 1957, the International Colloquium on Grafting was held in honor of the 100th anniversary of Daniel’s birth (Dean, 1962). The famous American horticulturist, Burbank, grafted a twig of a purple-leaved plum onto an old Kelsey plum tree. He was astonished when, from quantity of seeds gathered from the Kelsey plum tree, next season a seedling grew (among other plant) with deep purple leaves. He believed that the seedling was the result of graft hybridization between purple-leaved plum and Kelsey plum. There was no other purple-leaved plum within thousands of miles. The scion had not bloomed, and so crossing could not have occurred in the ordinary way (Burbank, 1914–1915).

C. Michurin’s contribution to graft hybridization In the course of his life, Michurin not only produced more than 300 varieties of horticultural plants but also made many observations and studies of plant life. The basic principle of Michurin’s operations was the changing of heredity by means of environmental changes acting on the early developmental stages of plants. He made much use of grafting as a means of influencing and improving immature plants. A key discovery was Michurin’s “mentor-grafting” method, with which Darwin’s “unknown conditions” of producing graft hybrids can be revealed. This method consists of the following—by grafting several scions taken from old varieties of fruit trees onto the lower branches of a young seedling’s crown, the young seedling acquires properties which it lacks; these properties are transmitted to the seedling through the grafted cuttings of the old varieties. That is why Michurin invoked the word “mentor” to describe this process. A stock plant could also used as a mentor—Michurin stated that a cutting from a young hybrid seedling of an apple, pear, or other kind of fruit tree, grafted onto the crown of an adult tree, could borrow the properties of the stock. He emphasized that this method can be employed effectively only on young seedlings, not on old and long-established varieties. By this method Michurin produced or improved several new varieties (Michurin, 1949). For example, Michurin grafted in 1894 a bud of a young Antonovka apple seedling to the crown of a 3-year-old pear wilding and obtained in 1898 an apple–pear graft hybrid with the pear-like shape of the fruit at the stem, which he named Reinette Bergamotte. In addition, Michurin provided a good answer to a question Darwin raised—as to why seeds obtained from cultivated fruit of apple and pear trees by natural pollination of flowers or by artificial hybridization produce—in most cases, when planted—an extremely high percentage of trees that yield fruit with undesirable properties. He proved that the main source of such “bad heredity” is the wild stock onto which the old cultivated variety was grafted. The scion itself—the old cultivated variety—is little changed by the action of the roots of

3. Plant Graft Hybridization

107

the wild stock; but its young organs (i.e., the seeds formed in the fruit), deviate strongly in the direction of the wild stock. This postulate is fully confirmed by the fact that seedlings with wild properties are never derived from the seeds of ungrafted trees of cultivated varieties if their blossoms are completely isolated from pollination by outside varieties. Michurin believed that the hereditary materials in the hybrid seedlings not only come from the crossed plants but also from the stocks onto which the crossed plants are grafted. In other words, the hybrid seedlings produced from grafted fruit trees are either sexual hybrids or graft hybrids. He thought that this is the main reason why Mendelian laws cannot be applied while crossing grafted fruit varieties. This is understandable because Mendel used annual and nongrafted pea plants and studied mainly qualitative characters, whereas Michurin used perennial and grafted fruit trees and studied mainly quantitative characters.

III. THE EXISTENCE OF GRAFT HYBRIDS A. Further evidence for graft hybrids It is clearly a matter of great interest to ascertain whether the formation of hybrids by grafting is really possible. In the Soviet Union, between 1938 and 1946, experiments were carried out by Khazina in which varieties of tomato were grafted onto other tomato varieties as well as onto species of other genera of Solanaceae (Morton, 1951). Most studies were those of I. E. Glushchenko during 1940s and 1980s (Glushchenko, 1946, 1958, 1962, 1973; Glushchenko et al., 1948, 1988). It can be estimated that there were about 500 papers on graft hybridization published in Soviet Union during 1950–1958 (Zu and Li, 1964). Unfortunately, these were largely thought by Western geneticists to involve fraudulent results (Crane, 1949; Hagemann, 2002). During recent decades, however, several independent groups of nonSoviet scientists repeatedly showed that graft-induced variant characteristics were stable and heritable. To take several examples: in 1955, Dr. Shinoto, the president of Japanese society of genetics, claimed to have obtained graft hybrids of eggplants: (1) The variety of Kantoao (blue fruits) was grafted onto the variety of Sinkuro (black fruits). Twenty were successfully grafted of which 16 bore fruits in the branches of the scions; in 9 plants the fruits were blue, while in the other 7 plants they were black. (2) Sinkuro was grafted onto Kantoao; out of four grafted plants two showed that the fruits on the stock were all blue, while in the other two plants bodies, leaves and fruits of the stocks all changed into black. (3) The first generation raised from seeds obtained by selfing in the blackened fruit borne on the scion of Kantoao showed segregation into the two types of plants: 25 blues and 10 blacks. It is worth mentioning that

108

Yongsheng Liu

similar positive results were obtained by Zu and Zhao (1957) in China, Stroun et al. (1963) in Switzerland, and Rajki and Pal (1966) in Hungary, in their experiments involving eggplant graft hybridization. Glavinic (1955) claimed to have heritably transmitted three singlegene Mendelian characters in tomato (cut leaf rather than potato leaf, yellow fruit rather than red, and short fruit rather than long) from the variety “Kartofelisn,” which was used as the stock, to the variety “GoldenTrophy,” which was used as scion. Scions were grafted as young seedlings at the cotyledonary stage. Significantly, it was the first-generation seedlings, produced from selfed fruits of the scion, and not the scion itself, that produced various combinations of stock and scion characters. When one plant of each combination was selfed, there was a tendency to breed true for the three single-gene Mendelian characters, although considerable segregation also occurred. Glavinic checked the homozygosity of these varieties for four generations before she started the grafting experiments. The buds on the scion were isolated by pergameneous bags, which were kept in place until they were removed by the fruit that had been formed in it. The parental Kartofelisni and Golden Trophy were grown as controls. Dean (1962) recognized that Glavinic’s work satisfied the minimal requirements for experiments presuming to prove the possibility of graft hybridization, although he was suspicious of Glushchenko’s claims. During a span of time between the 1950s and 1970s, Frankel demonstrated graft transfer of cytoplasmic male sterility in Petunia. Male-fertile maintainer scions were grafted onto male sterile stocks. Scion flower male-fertility remained autonomous, but male-sterile progeny were produced when scion flowers were selfed or crossed to other maintainer clones (Frankel, 1956, 1962, 1971). Edwardson and Corbett (1961) confirmed Frankel’s experimental claims. Attempted graft transfer of cytoplasmic male sterility has also been successful in sugar beets (Curtis, 1967) and alfalfa (Thompson and Axtell, 1978). Kasahara and his coworkers conducted extensively graft hybridization experiments in pepper (Capsicum annuum L.). In 1967, Ohta, a Japanese “Mendelist,” was allowed closely to examine Kasahara’s original data. He became aware that some anomalies could be attributed to mistakes but inferred that other incongruities seemed potentially real and beyond the knowledge of Mendelian genetics. He and his coworker carried out grafting experiments by using Kasahara’s materials and methods and produced variants similar to those of Kasahara’s. In their experiments, reciprocal grafts were made between two cultivars of red pepper: Tochigisantaka (with erect, fasciculate, and red fruits) and Kiiro (with pendent, nonfasciculate, and yellow fruits). An overall rate of variant occurrence of 0.84% was obtained in the first, second, and third generation self-crossed progeny of the scion (Ohta and Choung, 1975a). In another set of experiments Ohta and Choung (1975b) found that in stocks artificially infected with the single-stranded RNA virus for “broadbean wilt” the rate of

3. Plant Graft Hybridization

109

gene transfer increased dramatically from 2% for noninfected stocks to 16.5% for infected stocks. Apparently the hereditary changes associated with grafting were enhanced by virus infection. It should be noted that, unlike Kasahara, Ohta paid special attention to the characteristics of fruiting direction, fruiting position, and ripe pericarp color, since he knew that these were Mendelian traits with gene symbols already established; and they were clear enough to be understood by Mendelists, who had either refused to accept or been skeptical of Michurinists. From the beginning of his experiments, a special concern was to eliminate possible contamination at every step of the experiments. He used pure materials, which had been maintained for five generations by selfing, and sown the seeds in sterile soil and boxes. The genotype of each variant was determined by selfcrossing and testcrossing to both parents to check for the possibility of accidental contamination. Ohta and Chuong (1975a) concluded that contamination could not be responsible for the majority of the variants and that the rate of variant occurrence was too high for spontaneous mutation. Thus, as one citation of his paper noted—the work is thoroughly documented and there is no cause to doubt the genuineness of the results because these observations were made by professional Mendelian geneticists and have been repeated with similar results in independent laboratories (Pandey, 1985). Yagishita and coworkers have made clear the existence of graft hybrids in pepper, eggplant, and other plants (Hirata and Yagishita, 1986; Hirata et al., 2003; Taller et al., 1998, 1999; Yagishita and Hirata, 1986, 1987; Yagishita et al., 1990). In pepper, variant fruits were obtained from a “Yatsubusa” scion, which was grafted onto a “Spanish Paprika” stock, and had been stably inherited for at least 27 generations by seed propagation (Yagishita et al., 1990). Both the stock and the scion cultivars differed in many characteristics, and each repetition of grafting enhanced the range of variations in the variants (Taller et al., 1999). After the induction of genetic changes by repeated grafting, they selected variant lines based on variant fruit shape, and finally established stable graft hybrid lines, explained in part by invoking the term “G.” By using G5S45 generations (i.e., the 45th generation of sexual hybridization by inbreeding, after five successive grafting), they analyzed the graft-induced genetic changes and the inheritance of several characteristics (Taller et al., 1998). Now they are pursuing the possibility of foreign gene transport from stock to scion through the vascular system, integration into the genome, and sexual transmission to the scion progeny in graft system at molecular level (Hirata et al., 2003). Dole and Wilkins (1991) made auto and reciprocal grafts among different poinsettia cultivars. When the scions of Eckespoint C-1 Red (CR), a restricted-branching cultivar, were grafted onto the stocks of Annette Hegg Brilliant Diamond (BD), a free-branching cultivar, vegetative characteristics of branching pattern and leaf morphology of CR plants were altered when compared to the control graft combination CR/CR (scion/stock). Eckespoint C-1

110

Yongsheng Liu

Red scions grafted onto BD stocks produced a plant very similar to BD plants when axillary shoot length and node number were compared. However, axillary shoot diameter and leaf morphology were intermediate between CR and BD plants. Changes were retained after two generations of serial vegetative propagation and are considered permanent. In July 1965, Fan grafted a bud taken from an adult purple-leaved plum tree onto a 1-year-old apricot seedling. In March 1966, the main stem of the apricot seedling was wiped out, and then the bud of the purple-leave plum began to sprout and grow vigorously. In March 1980, surprisingly, an offshoot with purplish red leaves was produced from the root of the apricot and then was transplanted. The variant plant began to blossom in 1985. Interestingly, it closely resembled the scion of the purple-leaved plum but was totally different from its original plant—the stock of the apricot in characteristics (Fan, 1999). This provided evidence for the phenomenon underlying Michurin’s mentor-grafting method.

B. Reasons why graft hybrids have been met with skepticism Although the results, reviewed in an earlier section, seem to prove the existence of graft hybrids, the question arises as to why many biologists doubt their genuineness. Four factors account for this skepticism. First, many are evidently misled by the fact that, when old orchard plant varieties are propagated in the usual way by means of grafting, they hardly change at all under the influence of the wild stock. On this basis people imagine that the stock cannot influence any variety grafted onto it, even if the scion is at the juvenile stage. Botanists therefore suppose that graft propagation and graft hybridization are contradictory. For example, Soyfer (1994) recorded a most skeptical attitude about graft hybrids: “To me, the term is as much an oxymoron as “hot ice” or “dry water.” No claim that the term is “generally accepted” can be convincing, . . . since an error of logic should not be passed over in silence, regardless of who introduced the error.” Actually, graft propagation and graft hybridization differ with respect to the grafting method and the developmental stage of scion or stock. In graft hybridization, both mentor-grafting method and the method of producing chimera graft hybrid are very special grafting methods, which are totally different from the ordinary grafting methods applied in graft propagation. In addition, whether the scion or stock alters its hereditary traits depends on its developmental stage. Only the scions taken from young seedlings are susceptible to changes. It was a bitter lesson that made Michurin realize the difference between graft propagation and graft hybridization. Michurin very early conceived the idea of attempting to improve Russian fruit trees by introducing foreign materials into the country. He came first under the influence of Grell, a Moscow horticulturalist, who had developed the theory that superior foreign varieties could be acclimatized to the rigorous climate of Russia by being grafted

3. Plant Graft Hybridization

111

onto the hardy native stocks. Michurin worked along these lines for about 10 years but failed, finally rejecting the method as inefficacious, and realizing that only seeds and young seedlings that are plastic are susceptible to changes. According to Michurin, in the case of graft propagation the scion of the cultivated form, taken from an old tree, which has borne fruit for a number of years, possesses such a long developed power of stability that the young, and therefore weak, 2- or 3-year-old stock (wilding) is naturally unable to change. On the contrary, in the case of graft hybridization, when a scion taken from a young seedling is grafted onto the crown of an adult tree, the scion itself, as well as the seeds taken from the scion, will change its properties in the direction of the adult tree. Second, graft hybrids have been equated with graft chimeras. As mentioned at the beginning of this chapter, graft hybrid is defined as a type of plant chimera; this notion is rather one-sided because it ignores the existence of nonchimera graft hybrid (Winkler and Daniel’s “Influenced” graft hybrid as well as Michurin’s vegetative hybrid). It should be noted that the method of producing chimera graft hybrid is totally different from Michurin’s mentor-grafting method. Chimera graft hybrids are generated when an adventitious shoot apical meristem of multicellular origin arises from the graft union callus of a heterograft (Marcotrigiano, 2001), whereas nonchimera graft hybrids are generated when very young seedlings are grafted onto old plants or scions taken from old plants are grafted onto very young seedlings, in which young seedlings are susceptible to change in the direction of the old plants. The differences and similarities between chimera graft hybrid and nonchimera graft hybrid will be discussed later on. To avoid confusion, we can divide graft hybrids into two categories— chimera graft hybrid, which may be produced from graft union (see Fig. 3.1); and nonchimera graft hybrid, which may be produced by applying Michurin’s mentor-grafting method (Fig. 3.2). Third, some researchers obtained negative results in their experiments, so they denied the possibility of obtaining graft hybrids. For example, Sachs (1951) conducted experiments on intervarietal grafting in tomato, showing that there was no observable influence on leaf-shape or fruit-color either in the year of grafting or in the following generation. Stubbe (1954) carried out 2455 grafts between mutants, which were obtained by X irradiation of tomato varieties Condine Red and Lucullus, and between these mutants and the two original varieties, and concluded that no evidence to support the existence of graft hybrid was obtained. Kraevoi (1971) also claimed that he failed to obtain graft hybrids in his potato and tomato experiment. The question arises why some investigators, such as Sachs (1951), Stubbe (1954), and Kraevoi (1971), failed to obtain graft hybrids in their test experiments, whereas many others, such as Glavinic (1955), Glushchenko (1946), Frankel (1956, 1962, 1971), Ohta and Chuong (1975a,b), Shinoto (1955), Taller et al. (1998, 1999), and Yagishita and Hirata (1986, 1987), obtained graft hybrids in most cases. The answer is that it is

112

Yongsheng Liu

Figure 3.1. Diagram explaining the method of producing graft chimera—Winkler’s method. (A) Scion of nightshade is grafted onto the stock of tomato. (B) After union, the junction is cut transversely. (C) A shoot of sectorial chimera is produced from the junction.

not merely the grafting that counts but also the ability to skillfully apply the mentor-grafting method. I have carefully examined the grafting methods applied by Stubbe (1954) and Kraevoi (1971), and found that they did not follow mentor-grafting method properly. Although Sachs (1951) did apply mentorgrafting method in his tomato experiment, he only made observations in a short time. It is worth noting that successful outcomes have been rare in the short term; but most investigators working over periods of several years, were able to obtain graft hybrids. For example, Ohta (1961) obtained negative results when he studied graft transmission of cytoplasmic male sterility in red pepper. Later, however, he successfully obtained variant fruits in pepper (Ohta and Chuong, 1975a,b). Here I would like to tell a story pertaining to Zu’s grafting experiments, which were first done before 1945. At that time, he had only just heard about Michurin’s graft hybridization but had no idea how to proceed. The best he could do was to proceed based on his own imagination. Thus he tried twice, with negative results. But he did not give up hope because he believed that Michurin would not have presented anything in the way of falsified results. After 1949, Zu saw several references to Michurin’s doctrine, which prompted him to think about why his previous experiments failed. Zu concluded that he did not selectively use old and young plants and cut off the leaves of the plant

3. Plant Graft Hybridization

113

Figure 3.2. The most widely adopted mentor-grafting method in annual plants. (1) A seedling at cotyledonary phase is grafted onto an older stock. (2) Leaves of the scion except for two to three at the top are removed during the entire growth.

that expected to be influenced by the other plant. He subsequently obtained graft hybrids when he improved his grafting method (Zu and Zhao, 1957). Finally, the unknown mechanism of graft hybridization puzzles biologists. According to the chromosome theory of heredity, genes are located in the chromosome of the cell nuclei. Most people think that the scion and the stock could not have exchanged chromosomes of the cell nuclei; therefore, they do not think it possible to obtain hybrids by means of grafting. For example, Kraevoi (1971) claimed that true graft hybrids cannot be obtained and the cells of coexisting tissues of two different plants do not exchange genetic information. Now it is well known that mRNAs and small RNAs can move between the cells and around the plant (Lucas et al., 2001), and most biologists have accepted that certain genes routinely move around within a chromosome, between chromosomes, within a species, and between species (Comfort, 2001). In addition, some people attributed changes accompanying putative instances of graft hybridization to mutation or accidental pollination. But in most grafting experiments, the occurrence is as high as 10–15%, ostensibly far too high a value to be explained by mutations. The possibility of accidental fertilization by unknown pollen can also be rejected because in most grafting experiments the flower buds on the scion or stock were isolated in bags during the flowering period.

114

Yongsheng Liu

IV. METHODS OF GRAFT HYBRIDIZATION A. Production of chimera graft hybrids Accidentally produced chimeras have been observed to originate from adventitious shoots at graft unions. Winkler (1907) was the first to attempt to obtain chimera graft hybrid experimentally. He conducted his experiments by grafting shoots of the tomato onto the stem of the nightshade and vice versa (Fig. 3.1A). After union, the junction was cut through transversely (Fig. 3.1B). The surface thus exposed became quickly covered with callus derived partly from tissue of tomato and partly from that of nightshade. From this callus arose adventitious buds, which developed into shoots, the majority of which resembled either pure tomato or pure nightshade; but a few were interspecific chimeras. The initial interspecific chimeras were usually sectorial chimeras, the new shoot having arisen from both kinds of cells (Fig. 3.1C), but some eventually gave rise to stable periclinal chimeras, in which the core of one plant is covered by the skin of another (Burge et al., 2002; Neilson-Jones, 1969; Weiss, 1930). Production of chimera graft hybrids requires regeneration of shoots from the graft union after it has been cut back. An improved method named directionhormone slowly growing (DHS) for efficient introduction of disease resistance to chimera has been developed (Ohtsu, 1994). In addition, synthetic chimeras have been produced using in vitro graft techniques. Hirata et al. (1990) investigated in vitro graft culture methods that rely on the generation of adventitious shoots from cultured graft union tissue. After graft union, the grafted region was dissected into three parts (the fused apical part, a crosscut section of the graft union, and the united hypocotyls part) and subcultured on an MS medium containing auxins and cytokinins. Chimeric shoots were regenerated mainly from the crosscut section of the graft union near the apical meristem. They concluded that this technique was more effective than Winkler’s graft method (Burge et al., 2002; Noguchi et al., 1992).

B. Mentor-grafting procedures Mentor grafting involves a special kind of grafting method, which makes it possible for the characters and properties of younger plant seedlings to be partially altered in the direction of older plants. The pertinent procedures involve cuttings from an old established variety (mentor plant), grafted onto a young hybrid seedling (pupil plant); or, cuttings from young hybrid seedlings are grafted onto the crown of the old varieties. The mentor plant thus induces accelerated fruiting and deviation of the characteristics of the pupil plant in the desirable direction. The relevant material in which it is desired to produce changes—whether stock or scion—is taken at the younger stage. The younger the pupil plant is, the more successful is the experiment. By contrast, the mentor

3. Plant Graft Hybridization

115

plants must be older. After a graft has been accomplished it is desirable to remove the leaves as frequently as possible from the branches of the pupil plant. For the mentor plant, as many as possible of the leaves and branches should be preserved. The flower buds of the experimental plants should be carefully isolated in order to avoid accidental cross-pollination, and control plants should be raised each year to test their genetic purity and stability (Glushchenko, 1962; Michurin, 1949; Morton, 1951; Ohta, 1991). The most widely adopted mentor-grafting method involving annual plants consists of the following—very young seedlings (from the cotyledonary phase to three to five-leaved stage) are grafted onto mature stocks (2–3 months old, having 20–30 leaves). This ensures that the scions are fully dependent on the stock for nutrition. One-way flow of genetic material from stock to scion is affected by removing leaves of the scion (except for two to three at the top) twice a week during the entire time of growth. Usually it has been the progeny seedlings—produced from selfed fruits of the scion, and not the scion itself— that produced various combinations of stock and scion characters (Glavinic, 1955; Glushchenko, 1962; Ohta and Chuong, 1975a; Pandey, 1985).

V. CHARACTERISTICS OF GRAFT HYBRIDIZATION A. Systems based on chimera graft hybrids A chimera graft hybrid is an individual composed of genetically different cells and tissues, with the following characteristics: (1) mixed characters (Darwin, 1883; Glushchenko et al., 1948; Hirata et al., 1994). (2) Low-pollen fertility and seed-setting in interspecific and intergeneric chimera graft hybrids (Darwin, 1883; Glushchenko, 1973; Hirata et al., 1994). (3) Three kinds of such chimeras, based on spatial arrangement of the genetically distinct cells within these cell layers—sectorial chimeras in which a sector of all cell layers that is genetically different, periclinal chimeras in which one or more entire cell layer(s) is genetically distinct from another cell layer, and mericlinal chimera in which part of layer (or layers) is genetically different. Periclinal chimeras are the most stable form and can be multiplied by vegetative propagation (Burge et al., 2002; Marcotrigiano, 1997).

B. Systems based on nonchimera graft hybrids Experiments of this kind clearly revealed that grafting in annual plants (such as peppers and tomatoes) induce hereditary changes with the following characteristics: (1) Visible changes are rare in the year of grafting, but a small proportion of changes are observed in the first-seed generation, and these are inherited in the following generations (Glushchenko, 1958; Ohta, 1991).

116

Yongsheng Liu

(2) Changes occur at the gene level—in both directions, from dominant to recessive and recessive to dominant (Glushchenko, 1958; Hirata et al., 2003; Ohta, 1991). (3) Hereditary changes are only found in a comparatively small proportion of grafted plants; the overall rate of variant occurrence is approximately 1–22% in peppers (Ohta, 1991), 20% in eggplants (Rajki and Pal, 1966), and 10–15% in tomatoes (Glushchenko, 1953). (4) The changed traits are usually stable in the variant; but unstable mosaic forms are encountered more often among graft hybrids than among sexual hybrids (Glushchenko, 1958; Ohta, 1991). (5) Graft hybrids show certain characters of both graft components; in several of instances they also exhibit new characters (Glushchenko, 1958; Taller et al., 1998, 1999). The characteristics of graft hybridization in perennial fruit trees are as follows: (1) When both scion and stock are at adult stage, visible changes are very rare in either scion or stock, but a certain proportion of changes are observed in the seed progenies; and these are inherited in the following generations (Burbank, 1914–1915). (2) When the graft component, whether stock or scion, is a young seedling, visible changes can be observed frequently several years after grafting (Fan, 1999; Michurin, 1949), and these are inherited in the following generations and can also be multiplied by vegetative propagation (Morton, 1951).

VI. MECHANISMS UNDERLYING GRAFT HYBRIDIZATION A. Darwin’s Pangenesis, the first scientific explanation The first explanation offered for the formation of graft hybrid was Darwin’s Pangenesis. Simply put, Darwin proposed that cells are not only able to grow by means of cell division, but also are capable of “throwing off” minute particles or molecules, which he called gemmules. He assumed that all cells of the body throw off gemmules at various developmental stages. These gemmules are capable of self-replication, circulate in the body, and finally come to rest in the sex cells, or in parts where buds may be developed. In Darwin’s opinion, with regard to graft hybrids, gemmules released in the stock would be transferred into the scion and united with the sex cells and meristematic cells in the scion, resulting in heritable changes of the scion and their progenies. “On any ordinary theory of reproduction the formation of graft hybrids, and the action of the male element on the tissues of the mother plant, as well as on the future progeny of female animals, are great anomalies; but they are intelligible on our hypothesis” (Darwin, 1883). Many people thought that Darwin believed that the fusions of vegetative nuclei occurred at the graft union and resulted in graft hybrids (Burge et al., 2002; Marcotrigiano, 1997; Neilson-Jones, 1969; Tilney-Bassett, 1986). In fact

3. Plant Graft Hybridization

117

this is not the case. A careful reading of Darwin’s Variation book revealed that Darwin explained the formation of graft hybrids only with his Pangenesis.

B. Different viewpoints on the formation of graft hybrids Although Winkler was the first to coin the term chimera and to discover a simple way to produce chimeras by a special grafting technique, he recognized the existence of both chimera graft hybrids and nonchimera graft hybrids (Bailey, 1920). Unlike Winkler, Baur (1910) denied the existence of nonchimera graft hybrids and explained all the graft hybrids by his chimera hypothesis—that the chimeral pattern met with in the mature parts of certain plants is a development of pattern already present in the growing point (cited in Neilson-Jones, 1969; Tilney-Bassett, 1986). Baur (1910) assumes that there are cells of two different origins in the growing point. Thus in the periclinal chimeras, cells giving rise to the epidermis and those giving rise to the rest of the plant exist side by side, with no effect of one such tissue upon the other (cited in Swingle, 1927). In higher plants with stratified apices, the out apical cell layer has been termed “L1,” the layer subtending L1 has been termed “L2” and the layer beneath L2, if it exists, has been termed “L3” (Marcotrigiano, 2001). With few exceptions, in dicots the outer apical layer (L1) gives rise to the leaf epidermis; the second apical layer (L2) gives rise to gametes, the palisade parenchyma, the lower spongy parenchyma, and all of the spongy parenchyma of the leaf margin. The third apical layer (L3) gives rise to upper and middle layers of the spongy parenchyma (Marcotrigiano and Bernatzky, 1995). In recent years, however, observations made by many researchers have shown the presence of intermediate characters in chimera graft hybrids, and there is evidence that most parts of the chimera graft hybrids are influenced by both the parental genotypes (Byatt et al., 1977, Glushchenko et al., 1988; Hirata et al., 2001; Zhou et al., 2002). Gametes can also be derived from L1 or L3 (Hirata et al., 1994; Marcotrigiano and Bernatzky, 1995). Byatt et al. (1977) suggested that in the past too much emphasis has been laid on the anatomy of the epidermal layers and the separation of parental characters in chimera graft hybrids, and the chimera hypothesis needed to be modified to give more weight to the interactions between different cells and tissues. It has been also suggested that interactions between genetically different cells caused variations at the DNA level and thus could be a source of genetic variations (Glushchenko et al., 1988; Hirata et al., 2001; Zhou et al., 2002). With regard to the formation of nonchimera graft hybrids, Michurin (1949) believed that the genes might move between the stock and the scion. Later, however, the Soviet Michurinists explained the mechanism for graft hybridization by invoking “plastic substances.” In their opinion, the scion and the stock cannot exchange the chromosomes of the cell nuclei or the cytoplasm; yet, the hereditary properties can be transmitted from the stock to the scion and

118

Yongsheng Liu

vice versa. Therefore, plastic substances elaborated in the scion and in the stock possess the properties of the breed. “The plastic substances of the stock are an external element, food, with respect to the scion. Yet, these substances become, through assimilation, an integral part of the scion and alter its hereditary properties” (Morton, 1951). This hypothesis is rather soft because no indication was given as to what the “plastic substance” might be. It has been suggested since the 1970s that transformation is a probable mechanism for graft hybridization (Hirata et al., 2003; Ohta, 1991; Pandey, 1976; Taller et al., 1998, 1999). Ohta’s was the first to offer an explanation for graft transformation in pepper. In microhistological analysis of the stock stems he noticed that chromatin masses were moving through cell walls and intercellular spaces from the lignifying and dying cells toward the vascular bundles. He proposed that this chromatin must be transferred through the vascular system, across the graft union, to the floral primordial or growing points of the scion. In a normal plant no genetic effects of such chromatin movements would be noticeable because all cells have the same genotype. After grafting, however, particularly in certain Solanum plants, such as pepper, eggplant, and tomato, in which heavy lignification occurs in the stem, there may be considerable chromatin flow from stock to scion, especially when the scion is, in effect, parasitic on the stock (Ohta, 1991). This explanation seems reasonable, because several independent groups of Chinese investigators have demonstrated that, as a normal physiological phenomenon, not only metaplasm but also protoplasm translocates from old dying cells in lower withering leaves to the still active primordial; the latter can then be utilized as material for new cells (Cheng, 1956; Lou et al., 1957). Taller et al. (1998) found that some RAPD (random amplified polymorphic DNA) markers that were present in the graft-induced variants occurred in the stock, but were absent from the scion. The detection of stock DNA in the variants indicates that transfer of DNA from the stock to the scion’s gametes, and not mutation, is the cause of graft-induced changes. There is, however, no further cellular or molecular evidence revealing the possibility for, let alone the exact mechanism of, long-distance movement of DNA fragments in a given graft system (Zhang et al., 2002).

C. A new perspective on the mechanism of graft hybridization Stroun et al. (1977) described circulating nucleic acid in higher organisms. Several investigators have found that mRNA may move around a plant and among its cells. By grafting a piece of a cucumber onto a pumpkin plant, certain researchers showed that mRNA could move over long distances—in phloem sap from the cucumber graft, they detected pumpkin mRNA, indicating that these large molecules had traveled into the grafts (Xoconostle-Cazarers et al., 1999). Kim et al. (2001) showed that endogenous mRNA molecules in noninfected

3. Plant Graft Hybridization

119

plants not only travel between cells but also may execute their developmental functions in cells far removed from those in which the RNAs were transcribed. Grafts between tomato plants with normal and mutant leaf shapes showed that mRNA from the mutated gene, responsible for the altered leaf shape, could travel from the mutant stock into the wild-type scion and alter leaf shape in the otherwise genotypically wild-type scion. It is well known that retrotransposons are ubiquitous in plants and play a major role in plant gene and genome evolution, and in many cases, retrotransposons comprise 50% of nuclear DNA content (Kumar and Bennetzen, 1999). Retrotransposons are mobile genetic elements that transpose through reverse transcription of an RNA intermediate (Flavell, 1995; Kumar and Bennetzen, 1999). Retroviruses are also a potent vehicle for interspecies gene flow in plants (Peterson-Burch et al., 2000). With the discovery that novel mRNA species may move between cells and around the plant—and the ability of retroviruses or retrotransposons to reverse transcribe mRNA into cDNA capable of being integrated into the genome (Adler, 2001; Gabriel, 1998; Perlman and Boeke, 2004; Steele and Blanden, 2000)—one can surmise that mechanisms exist for horizontal gene transfer from stock to scion and vice versa. Darwin (1883) remarked that there is hardly a more amazing phenomenon in nature than the sensitivity of sex cells to external influences. Michurin (1949) emphasized repeatedly that the hereditary nature of young seedlings was profoundly affected by their environment. In plants, adaptive phenotypic plasticity is generally expressed for organisms exposed to an environmental perturbation during the early stages of development (Amzallag, 2004). In transgenic research, transformation and regeneration of woody fruit plants are usually limited to juvenile tissues derived from seeds or seedling organs, such as zygotic embryo, hypocotyls, or cotyledons; whereas tissue from mature plants cannot be readily transformed. In other cases—in which embryogenic cells of somatic origin, somatic embryos, or vegetatively propagated tissues have been used as starting material for transformation—the explants were juvenile or were rejuvenated by successive in vitro micropropagation (Cerera et al., 1998). That is, juvenile material has been extensively used for genetic transformation of woody fruit plants. It has been shown that the plants derived from the callus tissues are young in terms of their developmental stage and are biologically identical with those derived from seeds. In the practice of plant tissue culture, callus is susceptible to changes, and the shoots regenerated from callus usually have juvenile characters. In the practice of graft propagation, scions taken from adult cultivated varieties hardly change. Based on the facts just stated, Liu et al. (2004) postulated that germ cells, callus cells and, embryonic cells of the plants, as well as the somatic cells of the juvenile plants, are competent ones, which can be transformed easily by foreign genes; whereas somatic cells of the adult and old plants are noncompetent and difficult to transform by foreign genes.

120

Yongsheng Liu

I propose that the stock (or scion) mRNA molecules being transferred into the scion (or stock)—then reverse-transcribed into cDNA that can be integrated into the genome of the scion’s (or stock’s) germ cells, embryonic cells, callus cells, as well as the somatic cells of juvenile plants—may be the main mechanism for graft hybridization.

VII. SIGNIFICANCE OF GRAFT HYBRIDIZATION A. The phenomena considered with regard to plant genetics in general According to Darwin, it was the fact of graft hybridization and xenia that led him to formulate his suppositions about Pangenesis. Historical studies have shown that Darwin’s Pangenesis was one of the first really important theories of heredity and greatly influenced many subsequent theories, particularly those of Galton, Brooks, Weismann, and de Vries (Doncaster, 1912; Liu, 2005a). The word gene was coined in 1909 by Johanssen and was derived from de Vries’ term pangen (pangene), itself a derivative of the word pangenesis, which Darwin had coined. Pangenesis was largely thought to be flawed and wrong, not only because Weismann’s theory of germ-plasm forbids the inheritance of acquired characters, but also because there was no good evidence for graft hybridization as well as gemmule’s chemical existence. Although Weismann’s theory might apply to most or all animals, it cannot apply to plants because the sex cells of plants may arise from any tissue; and the sex organs are neither completely insulated, nor isolated from a plant’s “body” tissues. From this chapter, we learn that there is a considerable body of experimental evidence for the existence of graft hybrids. By comparing the nature and function of mRNAs with that of gemmules, Liu (2004a) has suggested their similarities to be striking and to provide actual evidence for the biochemical existence of gemmules. Once most geneticists have recognized the existence of graft hybrids and Darwin’s so-called gemmules, Pangenesis needs to be reconsidered. Plant chimeras have provided valuable investigative material to plant geneticists and physiologists. One important use of synthetic graft chimeras promoted studying the origin of tissues in different parts of the plant. In recent years, synthetic graft chimeras have also been used to study problems of incompatibility, to demonstrate the decisive role played by the epidermis in the perception of and response to light, and to assess stomatal responses to light (Tilney-Bassett, 1986). The generation and analysis of chimera graft hybrids have been used further to deduce patterns of cell division and cell fate during plant development and to demonstrate the existence of clonally distinct cell lineages in shoot meristems of higher plants (Szymkowiak, 1996).

3. Plant Graft Hybridization

121

Graft hybridization has played a crucial role in revealing the mystery of heredity in grafted fruit trees. More than 1400 years ago, Sixie Jia mentioned pear variability in the chapter “on grafting pear trees” of his book Qi Min Yao Shu: “There are about ten seeds in each fruit, but only two seedlings bear pear fruits with good quality, while the rest are useless, like the wild birchleaf pear (P. betulifolia)” (cited in Liu, 2000). This observation made at that time is still true today. According to Michurin’s observation, the overwhelming majority of hybrid seedlings, sometimes as high a proportion as 95%, have undesirable wild properties (Michurin, 1949). There was evidence that hybrid seedlings from cultivated pear varieties preponderate to wild type in fruit size and shape (Crane and Lewis, 1949). It has also been indicated that many apple varieties produce progenies in which the fruit size of the majority of the hybrid seedlings looks like wild type and only a very small proportion of hybrid seedlings produce fruit of acceptable size (Brown, 1960). Now most horticulturists have realized that the tendency of hybrid seedlings to a wild-like state is common. The question arises as to why the overwhelming majority of the hybrid seedlings that grow from the seeds gathered from grafted fruit varieties have undesirable wild properties. Michurin performed many studies that speak to this issue. In 1916, he wrote: “When we take a grafted tree of some varieties as the maternal parent in a cross, and of the resultant seedlings only a negligible proportion bear the characters of the parent types, while the majority are simply wildings, the cause is not atavism at all, but almost exclusively the very strong and stable influence of the maternal plant’s stock upon the extremely weak and unstabilized constitution of the hybrid seeds. In other words, what we get is graft hybrids of the wild stock, with only the most negligible admixture of the properties of the cultivated varieties” (Michurin, 1949) This is the reason why the heredity of grafted fruit trees violates Mendel’s laws of heredity. It is worth noting that, unlike his followers, Michurin did not deny the value of Mendelian laws. He wrote: “I by no means deny the merits of Mendelian laws. On the contrary, I merely insist on the need to introduce amendments and addenda into it, for it is evident to everybody that his calculations are not applicable to cultivated varieties of fruiters . . . .” (Michurin, 1949)

122

Yongsheng Liu

B. The phenomenon considered from the perspective of plant breeding In the field of plant improvement, graft hybridization may provide simpler alternatives to in vitro transformation, mutagenesis, somatic cell hybridization, and regeneration in tissue culture. While the present techniques of DNA engineering offer exciting possibilities for genetic manipulation of qualitative characters controlled by one or a few specific genes, they offer no similar solution, in the foreseeable future, for quantitative characters that are controlled by large numbers of genes. Many of the important characters of economically significant plants are of the latter type (Pandey, 1985). Importantly, graft hybridization may affect quantitative characters either alone or in combination with qualitative characters. Moreover, the techniques applied to affect such hybridizations not only may reduce the time needed to produce a given new variety, but also, by breaking down linkage relationships, may allow selective transfer of desirable genes alone from relatively distantly related species (Liu, 2000; Liu et al., 2004; Pandey, 1976). Mentor-grafting methodology has been proved to provide a powerful means of plant breeding. By applying such methods, workers in the Soviet Union created many new fruit, crop, and vegetable varieties with excellent quality and high yields (Glushchenko, 1962; Michurin, 1949; Morton, 1951). Japanese researchers demonstrated that new characters induced by grafting are stable traits and can be used as novel genetic source material. These investigators produced five new variant lines and several sublines by using the graft-induced variant strain and other two cultivars (Taller et al., 1999). In addition, many new crop varieties have been produced by means of grafting in China (Liu et al., 2004; Zhang et al., 2002). The unique characteristics of periclinal chimeras show the potential of chimeral breeding to produce new cultivars. Many horticulturally important cultivars are chimera graft hybrids, especially periclinal chimeras, which can be propagated vegetatively (Burge et al., 2002). Therefore, the synthesis of chimeras offers a novel breeding approach for producing plants combining valuable characteristics from two different cultivars, such as disease or insect resistance in the epidermis, with the desirable core tissue characteristics of an otherwise susceptible cultivar. In this respect, Clayberg (1975) produced a periclinal chimera with an epidermis of Solanum pennelli and a core of tomato. The tomato strain was susceptible, and S. pennelli highly resistant, to both greenhouse whitefly and potato aphid. The chimera was highly resistant to whitefly and susceptible to potato aphid. Ohtsu (1994) suggested that introducing tissue of a disease-resistant cultivar to the second- and third-germ layers, and tissue of a high-quality cultivar to the first layer, would make a chimeric tree

3. Plant Graft Hybridization

123

with disease resistance to canker and Citrus tristeza closterovirus, which would retain the obviously desirable property of producing high-quality fruit. Graft hybridization plays an important role in guiding parental selection in situations involving sexual hybridization associated with fruit-tree breeding. It was Michurin who first noticed that all fruit plants that are not grafted, but have their own roots, and when crossed give a greater number of cultivated varieties with good qualities, compared with situations in which plants are grafted onto wild stock. This clearly shows that the plant’s root system plays an active part in the formation of seeds. Based on these findings, Michurin (1949) warned breeders that the crossed parental plants should be ones that are not grafted but are standing on their own roots; in this way the numbers of new varieties with good qualities would increase fourfold. Further understanding of the mechanisms underlying graft hybridization can correct mistaken measures adopted by some plant breeders. Because of the long generation times of fruit trees, there has been much interest in shortening the breeding cycle. To save time, many workers used to graft young hybrid seedling onto limbs of a fruited tree. Although this method can effectively shorten the juvenile phase, it usually results in deterioration of fruit qualities (Michurin, 1949). It has been turned out that the deterioration of new variety in such cases is due to the influence of the wild stock on which the adult tree of the cultivated type has been grafted and grown (Michurin, 1949). For what one obtains by such means is not pure hybrids resulting from a cross, but graft hybrids between the scion and the stock. To solve this problem, Michurin (1949) proposed a new method: by taking three or four cuttings from an excellent variety—and grafting them onto the lower branches of this hybrid seedling’s crown, not far from the trunk—the hybrid seedling will, under the influence of such a mentor, bear fruit within the next 2 years. After this, the mentor scions must be cut off; otherwise the mentor variety’s influence may also affect the hybrid seedling’s characters. In this way the breeding cycle can be shortened, and the deterioration of fruit qualities can also be avoided. Michurin was one of the first investigators in the history of plant breeding to use not only interspecific, but also intergeneric hybridization between taxonomically remote species. He thus obtained dozens of valuable varieties of fruit and soft-fruit crops and of ornamental as well as other plants. In hybridizations involving distantly related species, it is necessary to find ways and means to overcome the resistance to crossing. Michurin’s method of “preliminary vegetative approximation,” which combines graft hybridization and sexual hybridization, has been widely applied not only to fruit trees, but also to annual plants. In this regard, it is well known that wheat and rye are very difficult to hybridization. Hall (1954) found that only 2–3% of wheat flowers

124

Yongsheng Liu

pollinated by rye gave seeds. But of 2897 wheat flowers borne by plants—which, as embryos, had been grafted onto rye endosperm—400 (14%) gave seeds when pollinated by rye. It has been shown with statistical certainty that the crossability between wheat and rye can be increased if the wheat plants used for the crosses are derived from embryos—those that were grafted onto rye endosperm.

VIII. CONCLUSIONS In the history of genetics, neglect of certain findings and phenomena is not uncommon. It is well known that Mendel’s laws of heredity and McClintock’s work on transposable elements were neglected for decades. Less well known is that Darwin (1883) and Michurin’s (1949) work on graft hybridization, which was largely ignored for about a century. Nevertheless, the “ignored” case at hand in the discussion involves extensive documentation of graft hybridization. I conclude that there is no cause to doubt the existence of graft hybrids. To avoid confusion, we need to distinguish between graft propagation and graft hybridization, which are affected by the grafting method and by the stage of plant development. It also should be noted that chimera graft hybrids comprise but one type of graft hybrid. Further studies will be required to elucidate molecular mechanisms underlying graft hybridization. In this respect, heterografting experiments have demonstrated that mRNAs and small RNAs are transported between the stock and the scion in the network of phloem tubes that carry sap (Flintoft, 2004). However, there is as yet no further cellular or molecular evidence pointing toward the possibility of long-distance movement of DNA fragments in the graft system, let alone the mechanism by which this would occur. In animals, it has been proved that substantial amounts of degraded genomic DNA are present in blood plasma and tissue fluids, derived from cells in the body that died. This circulating DNA binds to receptors on the surface of living cells and is taken up and transported to the cell nucleus (Yakubov et al., 2002). Could there be “circulating DNA” in sap—a plant’s “blood”? To proceed with this thought, recall that relatively young plants are susceptible to change under the influence of a given stock or scion plant. In this context, a study showed that traffic of macromolecules (including proteins and nucleic acids) between plant cells is most promiscuous in young, undifferentiated tissues, becoming much more restricted as tissues mature. The younger they are, the larger their plasmodesmata (Ueki and Citovsky, 2005). It has been suggested that graft hybridization is striking evidence in favor of Darwin’s Pangenesis, which has been largely thought to be wrong and is now only of historical interest. Mendelian laws, as any fruitful theory, have led to predictions of many new findings. But it certainly was not obvious that it also

3. Plant Graft Hybridization

125

could provide scientific explanations for a considerable variety of phenomena including graft hybridization. It still pays to go back to Darwin’s Pangenesis not only for historical reasons but also because in many respects it is surprisingly modern. In an issue of Nature, Lolle et al. (2005) reported that Arabidopsis plants homozygous for recessive mutant alleles of the organ fusion gene can inherit allele-specific DNA sequence information that was not present in the chromosomal genome of their parents but was present in previous generations. To explain this interesting reversion phenomenon, they proposed a model in which a type of stable RNA can be replicated and transmitted over multiple generations. This proposal parallels Darwin’s theory of Pangenesis (Liu, 2005b). Graft hybridization is not only a simple and powerful means of plant breeding, but also plays a crucial role in revealing the mystery of non-Mendelian inheritance in grafted fruit trees. The breeding of new varieties of such trees is important from the standpoints of human nutrition and economics. Until now, however, our ability to manipulate fruit-tree varieties through classic genetics has been limited because Mendelian laws cannot be applied while crossing grafted fruit varieties. The time has come when further progress in our understanding of graft hybridization requires that we reconsider Michurin’s principles and methods in plant breeding.

Acknowledgments I am deeply indebted to Dr. Anne McLaren for her numerous suggestions and constant encouragement. This chapter could not have been written without her inspiration, help, and advice. I am also grateful to Dr. Jeffrey C. Hall and other reviewers for their important suggestions and corrections.

REFERENCES Adler, M. J. (2001). A novel mechanism for evolution? Science 294, 53–54. Amzallag, G. N. (2004). Critical periods as fundamental events in life. Theor. Biosci. 123, 17–32. Bailey, L. H. (1920). “Plant Breeding.” The Macmilian Company, New York. Baur, E. (1910). Propfbastarde. Biologische Zentralblatt 30, 497–514. Brown, A. G. (1960). The inheritance of shape, size and season of ripening in progenies of the cultivated apple. Euphytica 9, 327–337. Burbank, L. (1914). “His Methods and Discoveries and Their Practical Application.” Luther Burbank Press, New York. Burge, G. K., Morgan, E. R., and Seelye, J. F. (2002). Opportunities for synthetic plant chimeral breeding: Past and future. Plant Cell, Tissue Organ Cult. 70, 13–21. Byatt, J. I., Ferguson, I. K., and Murray, B. G. (1977). Intergeneric hybrids between Crataegus L. and Mespilus L.: A fresh look at an old problem. Botanical Journal of the Linnean Society 74(4), 329–343. Cerera, M., Juarez, J., Navarro, A., Pina, A. J., Nuran-Vila, N., Navarro, L., and Pena, L. (1998). Genetic transformation and regeneration of mature tissues of woody fruit plants bypassing the juvenile stage. Transgenic Res. 7, 51–59.

126

Yongsheng Liu

Cheng, K. C. (1956). On the process of the intercellular migration of chromatin substance and new formation of nucleus in the pollen mother cells of Lilium Sutchuenense Franch. Scientia Sinica 5, 497–507. Clayberg, C. D. (1975). Insect resistance in a graft-induced periclinal chimera of tomato. Hortscience 10, 13–14. Comfort, N. C. (2001). Are genes real? Nat. Hist. 110(5), 28–35. Crane, M. B. (1949). Lysenko on grafting and genetics. Discovery 10, 64–66. Crane, M. B., and Lewis, D. (1949). Genetical studies in pears. Vegetative and fruit characters. Heredity 3, 85–97. Curtis, G. J. (1967). Graft-transmission of male sterility in sugar beet (Beta Vulgaris L.). Euphytica 16, 419–424. Daniel, L. (1929). The inheritance of acquired characters in grafted plants. In “Proceedings of the International Congress of Plant Sciences” (B. M. Duggar, ed.), pp. 1024–1044. George Banta Publishing Company, Menasha, WI. Darwin, C. (1883). “The Variation of Animals and Plants Under Domestication.” D. Appleton & Co., New York. Dean, D. S. (1962). An evaluation of the claims of I. E. Glouchtchenko for vegetative hybridization in plants. J. Hered. 53, 215–221. Dole, J. M., and Wilkins, H. F. (1991). Vegetative and reproductive characteristics of poinsettia altered by a graft-transmissible agent. J. Am. Hort. Soc. Sci. 116, 307–311. Doncaster, L. (1912). “Heredity In the Light of Recent Research.” Cambridge University Press, Cambridge. Edwardson, J. R., and Corbett, M. K. (1961). Asexual transmission of cytoplasmic male sterility. Proc. Nat. Acad. Sci. 47, 390–396. Fan, S.-Y. (1999). Phenotype variation by the action of scion Prunus japonica Thunb on stock Prunus armeniaca L. Hereditas (Beijing) 21(4), 43–44. Flegr, J. (2002). Was Lysenko (partly) right? Michurin biology in the view of modern plant physiology and genetics. Riv. Biol./Biol. Forum 95, 259–272. Flavell, A. J. (1995). Retroelements, reverse transcriptase and evolution. Comp. Biochem. Physiol. 110(B), 3–15. Flintoft, L. (2004). Small RNAs take the tube. Nat. Rev. Genet. 5, 1038. Frankel, R. (1956). Graft-induced transmission to progeny of cytoplasmic male sterility in petunia. Science 124, 684–685. Frankel, R. (1962). Further evidence on graft-induced transmission to progeny of cytoplasmic male sterility in petunia. Genetics 47, 641–646. Frankel, R. (1971). Genetic evidence on alternative maternal and Mendelian heredity element in petunia. Heredity 26, 107–119. Gabriel, A. (1998). LCMV cDNA formation: Which reverse transcriptase is responsible? Trends Genet. 14, 220–221. Glavinic, R. (1955). Vegetative hybridization of tomato. Proc. 14th. Int. Hort. Congr. 1, 440–445. Glushchenko, I. E. (1946). Experimental data on hybridizing tomatoes by grafting. Agrobiologija (Agrobiology) (3), 78–105. Glushchenko, I. E., Bazavluk, V. J., and Medvedeva, G. B. (1948). Plant chimeras as a form of vegetative hybrids. Trudy Inst. Genetiki (Proc. Inst. Genetics) 15, 58–97. Glushchenko, I. E. (1953). “The Significance of Vegetative Hybridization in Plant Genetics” (in chinese, translated by X. -L. You). The Chinese Academy of Sciences, Beijing. Glushchenko, I. E. (1958). Some regularities of vegetative hybridization of plants. Proc. 10th Int. Congr. Genet. (Montreal) 2, 333. Glushchenko, I. E. (1962). Vegetative hybridization and its importance for plant breeding. Trud. Inst. Genet. Trudy Inst. Genetiki (Proc. Inst. Genetics) 29, 40–66.

3. Plant Graft Hybridization

127

Glushchenko, I. E. (1973). The nature of chimerical plants. Plant Breeding Abstract 43(6), 335. Glushchenko, I. E., Nedesheva, G. N., and Avetisova, L. V. (1988). Cell engineering in vivo in some vegetable crops. Plant Breeding Abstracts 58(7), 670–671. Hagemann, R. (2002). How did East German genetics avoid Lysenkoism? Trends Genet. 18, 320–324. Hall, O. L. (1954). Hybridization of wheat and rye after embryo transplantation. Hereditas 40, 453–458. Hartmann, H. T., Kester, D. E., Davies, F. T., and Geneve, R. L. (1997). “Plant Propagation: Principles and Practices.” Prentice Hall, Upper Saddle River, NJ. Hirata, Y., and Yagishita, N. (1986). Graft-induced changes in soybean storage proteins. I. appearance of the changes. Euphytica 35, 395–401. Hirata, Y., Yagishita, N., Sugimoto, M., and Yamamoto, K. (1990). Intervarietal chimera formation in cabbage (Brassica oleracea L.). Jpn. J. Breed 40, 419–428. Hirata, Y., Noguchi, T., Oguni, S., and Kan, T. (1994). Genetic constitution of germ cells in intervarietal and interspecific chimeras of Brassica induced by in vitro grafting. Theor. Appl. Genet. 89, 249–264. Hirata, Y., Takeda, Y., and Motegi, T. (2001). Induction of cytoplasmic male sterility in the progeny derived from interspecific chimera between Brassica oleracea and B. campestris. Euphytica 117, 143–149. Hirata, Y., Ogata, S., Kurita, S., Nozawa, G. T., Zhou, J., and Wu, S. (2003). Molecular mechanism of graft transformation in Capsicum annuum L. Acta Hort. 625, 125–130. Jouin, M. E. (1900). Can hybrids be obtained by grafting? J. R. Hort. Soc. 24, 237–240. Kasahara, T., Nakamura, T., and Yoneyama, Y. (1973). Graft-induced variation in red peppers (Japanese). Recent Advances in Breeding 13, 73–89. Kim, M., Canio, W., Kessler, S., and Sinha, N. (2001). Developmental changes due to long-distance movement of homeobox fusion transcript in tomato. Science 293, 287–289. Kraevoi, S. Y. (1971). The possibilities of using grafting in plant breeding. In “Genet. Osnovy Selektsii Rast” (N. P. Dubinin, ed.), pp. 343–373. Nauka, Moscow. Kumar, A., and Bennetzen, J. L. (1999). Plant retrotransposons. Annu. Rev. Genet. 33, 479–532. Landman, O. E. (1991). The inheritance of acquired characteristics. Annu. Rev. Genet. 25, 1–20. Landman, O. E. (1993). Inheritance of acquired characteristics revisited. Bioscience 43, 696–705. Liu, Y.-S. (2000). The records of plant graft hybridization in ancient China. In “Agricultural History” (Z.-J. Zhou and G.-J. Ni, eds.), pp. 308–317. Jiangxi People’s Press, Nanchang. Liu, Y.-S. (2004a). Further evidence for Darwin’s pangenesis. Riv. Biol./Biol. Forum 97, 53–66. Liu, Y.-S. (2004b). Lysenko’s contributions to biology and his tragedies. Riv. Biol./Biol. Forum 97, 483–498. Liu, Y.-S., Li, B.-Y., Li, G.-R., and Zhou, X.-M. (2004). Graft hybridization and the specificity of heredity in fruit trees. Hereditas (Beijing) 26(5), 705–710. Liu, Y.-S. (2005a). Darwin and Mendel: Who was the pioneer of genetics? Riv Biol/Biol Forum 98, 305–322. Liu, Y.-S. (2005b). Reversion: Going back to Darwin’s works. Trends Plant Sci. 10(10), 459–460. Lolle, S. J., Victor, J. L., Young, J. M., and Pruitt, R. E. (2005). Genome-wide non-Mendelian of extra-genomic in formation in. Arabidopsis. Nature 434, 505–509. Lou, C. H., Wu, S. H., Chang, W. C., and Shao, L. M. (1957). Intercellular movement of protoplasm as a means of translocation of organic material in garlic. Scientia Sinica 6, 139–157. Lucas, W. J., Yoo, B.-C., and Kragler, F. (2001). RNA as a long-distance information macromolecule in plants. Nat. Rev. Mol. Biol. 2, 849–857. Marcotrigiano, M., and Bernatzky, R. (1995). Arrangement of cell layers in the shoot apical meristem of periclinal chimeras influences cell fate. Plant J. 7, 193–202. Marcotrigiano, M. (1997). Chimeras and variegation: Patterns of deceit. Hortscience 32, 773–784.

128

Yongsheng Liu

Marcotrigiano, M. (2001). Genetic mosaics and the analysis of leaf development. Int. J. Plant Sci. 162(3), 513–525. Martin, E., and Hine, R. S. (2000). “A Dictionary of Biology.” Oxford University Press, Oxford. Michie, D. (1958). The third stage in genetics. In “A Century of Darwin” (S. S. Barnett, ed.), pp. 56–84. Heinemann, London. Michurin, I. V. (1949). “Selected Works.” Foreign Languages Publishing House, Moscow. Morton, A. G. (1951). “Soviet Genetics.” Lawrence & Wishart, London. Neilson-Jones, W. (1969). “Plant Chimeras,” 2nd Edn. Methuen, London. Noguchi, T., Hirata, Y., and Yagishita, N. (1992). Intervarietal and interspecific chimera formation by in vitro graft-culture method in Brassica. Theor. Allp. Genet. 83, 727–732. Ohta, Y. (1961). Grafting and cytoplasmic male sterility in. Capsicum. Seiken Ziko 12, 35–43. Ohta, Y., and Chuong, P. V. (1975a). Heredity changes in Capsicum annuum L. I. Induced by ordinary grafting. Euphytica 24, 355–368. Ohta, Y., and Chuong, P. V. (1975b). Heredity changes in Capsicum annuum L. II. Induced by virusinoculated grafting. Euphytica 24, 605–611. Ohta, Y. (1991). Graft-transformation, the mechanism for graft-induced genetic changes in higher plants. Euphytica 55, 91–99. Ohtsu, Y. (1994). Efficient production of a synthetic periclinal chimera of citrus “NF-5” for introduction of disease resistance. Ann. Phytopath. Soc. Jpn. 60, 82–88. Pandey, K. K. (1976). Genetic transformation and graft hybridization in higher plants. Theor. Appl. Genet. 47, 299–302. Pandey, K. K. (1985). Gene movement and genetic manipulation. In “Plant Genetics” (M. Freeling, ed.), pp. 235–249. Alan R. Liss, Inc, New York. Perlman, P. S., and Boeke, J. D. (2004). Ring around the retroelement. Science 303, 182–184. Peterson-Burch, B. D., Wright, D. A., and Laten, H. M. (2000). Retroviruses in plants? Trends Genet. 16, 151–152. Rajki, E., and Pal, G. (1966). Transplantation of a part of the berry fruit of Solanum melongena L. Acta Agron. Acad. Sci. 15(3–4), 301–309. Redei, G. P. (1998). “Genetics Manual: Current Theory, Concepts, Terms.” World Scientific, Singapore. Sachs, L. (1951). “Vegetative hybridization” in the tomato. Nature 167, 282–283. Shinoto, Y. (1955). Graft experiments in eggplant. Kagaku 25, 602–607. Soyfer, V. N. (1994). “Lysenko and the Tragedy of Soviet Science.” Rutgers University Press, New Brunswick. Steele, E. J., and Blanden, R. V. (2000). Lamarck and antibody genes. Science 288, 2318–2319. Stroun, M., Mathon, C. C., and Stroun, J. (1963). Modifications transmitted to the offspring, provoked by heterograft in Solanum Melongena. Arch. Sci. 16, 225–245. Stroun, M., Anker, P., Maurice, P., and Gahan, P. B. (1977). Circulating nucleic acid in higher organisms. Int. Rev. Cytol. 51, 1–48. Stubbe, H. (1954). On vegetative hybridization in plants: Experiments with tomato mutants. Kulturpflanze 2, 185–236. Swingle, C. F. (1927). Graft hybrids in plants. J. Hered. 18, 73–94. Szymkowiak, E. J. (1996). What chimeras can tell us about plant development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 351–376. Taller, J., Hirata, Y., Yagishita, N., Kita, M., and Ogata, S. (1998). Graft-induced changes and the inheritance of several characteristics in pepper (Capsicum annuum L.). Theor. Appl. Genet. 97, 705–713. Taller, J., Yagishita, N., and Hirata, Y. (1999). Graft-induced variants as a source of novel characteristics in the breeding of pepper (Capsicum annuum L.). Euphytica 108, 73–78.

3. Plant Graft Hybridization

129

Thompson, T. E., and Axtell, J. D. (1978). Graft-induced transmission of cytoplasmic male sterility in alfalfa. J. Hered. 69, 159–164. Tilney-Bassett, R. A. E. (1986). “Plant Chimeras.” Edeard Arnold, London. Ueki, S., and Citovsky, V. (2005). Control improves with age: Intercellular transport in plant embryos and adults. Proc. Natl. Acad. Sci. USA 102, 1817–1818. Weiss, F. E. (1930). The problem of graft hybrids and chimaeras. Biol. Rev. 5, 231–271. Winkler, H. (1907). Description of S. nigrolycopersicum and method of obtaining graft hybrids. Ber. Deut. Bot. Ges. 25, 568–576. Xoconostle-Cazarers, B., Xiang, Y., Ruiz-Medrano, R., Wang, H. L., Monzer, J., Yoo, B. C., Mcfarland, K. C., Franceschi, V. R., and Lucas, W. J. (1999). Plant paralog to viral movement protein that potentiates transport of mRNA into the phloem. Science 283, 94–98. Yagishita, N., and Hirata, Y. (1986). Genetic nature of bushy plant type in the variant strain induced by grafting in pepper. Euphytica 35, 17–23. Yagishita, N., and Hirata, Y. (1987). Graft-induced changes in fruit shape in pepper. I. Genetic analysis by crossing. Euphytica 36, 809–814. Yagishita, N., Hirata, Y., Mizukami, H., Ohashi, H., and Yamashita, K. (1990). Genetic nature of low capsaicin content in the variant strains induced by grafting. Euphytica 46, 249–252. Yakubov, L. A., Petrova, N. A., Popova, N. A., Semenov, D. V., Nikolin, V. P., and Os’kina, I. N. (2002). The role of extracellular DNA in the stability and variability of cell genomes. Dokl. Biochem. Biophys. 382, 31–34. Zhang, D.-H., Meng, Z.-H., Xiao, W.-M., Wang, X.-C., and Sodmergon (2002). Graft-induced inheritable variation in mungbean and its application in mungbean breeding. Acta Bot. Sin. 44, 832–837. Zhou, J.-M., Hirata, Y., Nou, I.-S., Shiotani, H., and Ito, T. (2002). Interactions between different genotypic tissue in Citrus graft chimeras. Euphytica 126, 355–364. Zhou, Z.-J. (1994). Origin and development of grafting in ancient China. Stud. History Nat. Sci. 13(3), 264–272. Zu, D.-M., and Zhao, Y.-S. (1957). A study on the vegetative hybridization of some solanaceous plants. Scientia Sinica 6, 889–903. Zu, D.-M., and Li, J.-G. (1964). “Vegetative Hybridization in Plants and Animals.” Science Press, Beijing.

This page intentionally left blank

4 Step into the Groove:

Engineered Transcription Factors as Modulators of Gene Expression Astrid E. Visser,* Pernette J. Verschure,† Willemijn M. Gommans,‡ Hidde J. Haisma,‡ and Marianne G. Rots‡,§§Corresponding author: [email protected] *Department of Molecular Genetics, Leiden Institute of Chemistry University of Leiden, 2300 RA Leiden, The Netherlands † Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, 1098SM Amsterdam, The Netherlands ‡ Therapeutic Gene Modulation Groningen University Institute for Drug Exploration University of Groningen, The Netherlands

I. Introduction II. Transcriptional Therapy A. Epigenetic therapy B. Posttranscriptional gene silencing C. Gene therapy D. Sequence-specific DNA-binding proteins as engineered transcription factors III. Engineered Zinc-Finger–Based Transcription Factors (ZF-TFs) and the Influence of Nucleosomes A. ZFP binding and nucleosomal structure B. Target definition IV. Concluding Remarks and Future Perspectives Acknowledgments References

§

Corresponding author: [email protected]

Advances in Genetics, Vol. 56 Copyright 2006, Elsevier Inc. All rights reserved.

0065-2660/06 $35.00 DOI: 10.1016/S0065-2660(06)56004-3

132

Visser et al.

ABSTRACT Increasing knowledge about the influence of dysregulated gene expression in causing numerous diseases opens up new possibilities for the development of innovative therapeutics. In this chapter, we first describe different mechanisms of misregulated gene expression resulting in various pathophysiological conditions. Then, an overview is given of different technologies developed to readjust expression levels of genes. One of the most promising upcoming approaches in this respect is the development of engineered zinc-finger transcription factors. Results obtained from modulating endogenous gene expression using such engineered transcription factors are reviewed in depth. Finally, we address possible pitfalls of using such transcriptional targeting approaches at the “chromatin level.” We describe aspects of studies at this level that influence successful DNA binding of engineered transcription factors, thereby affecting gene activity. Engineered transcription factors have great promise as potent therapeutics. Moreover, this technology is expected to yield fundamental knowledge about the organization and function of the genome. ß 2006, Elsevier Inc.

I. INTRODUCTION An increasing number of diseases is linked to defects at the level of DNA. Often such pathologies represent as a mutation in the protein-coding region, but dysregulated gene expression is receiving growing interest for its association with cellular dysfunction. Gene expression is a tightly regulated process involving opening up of the chromatin structure and binding of transcription factors to regulatory DNA sequences. We here describe the main mechanisms resulting in disrupted gene expression, which can be divided into phenomena involving epigenetic processes, chromosomal rearrangements, and mutations in transcription factor coding regions as well as mutations in their binding sites. Clinical examples of disruptions in such mechanisms are reviewed in detail elsewhere (Arnaud and Feil, 2005; Ausio et al., 2003; Kleinjan and van Heyningen, 2005; Villard, 2004). Clinical phenotypes that are caused by disturbed gene expression have been assigned to abnormalities at the chromatin structure (so-called epigenetic profiles (Egger et al., 2004), see also accompanying chapter (Verschure et al., Chapter 5, this issue). Three main systems are of importance in causing epigenetic abnormalities—DNA methylation, histone modifications, and RNA-associated gene silencing. DNA methylation plays an important role in silencing of human genes. Although methylation takes place at the level of the DNA (cytosine), this modification does not alter the DNA sequence and is reversible. It is therefore considered an epigenetic modification. In a substantial

4. Successes and Pitfalls of Engineered Transcription Factors

133

number of Beckwith-Wiedemann patients the expression of the normally imprinted H19 (maternally active) and IGF2 (paternally active) genes are disrupted due to mutations affecting the DNA methylation status of the upstream region of the H19 gene (O’Neill, 2005; Thorvaldsen et al., 1998). Also in various cancer types, many important genes have been described to be hypermethylated (Gilbert et al., 2004). Histone modifications, such as acetylation or methylation, are markers for transcriptionally competent or incompetent chromatin. Mutations in a histone acetyltransferase, encoded by crebbp, for example, have been associated with the Rubinstein-Taybi retardation (Ausio et al., 2003). In addition to DNA methylation and histone modifications, it has been shown that small RNA molecules contribute to heritable gene silencing by inducing methylation of promoter areas (O’Neill, 2005). Chromosomal rearrangements or deletions can be involved in causing diseases by physically disturbing the long-range regulation of gene expression. In certain forms of deafness, for example, deletion in regulatory sequences of the gene encoding the POU3F4 transcription factor have been identified, which are located as far as 900 kilobase pairs away from the transcription initiation site (de Kok et al., 1995). Also, effects of changed gene expression levels can be caused by an altered chromatin organization associated with the new location of the gene in a particular region of the cell nucleus, which is described in the accompanying chapter (Verschure et al., Chapter 5, this issue). Most diseases that are due to dysregulated long range control show disrupted expression of genes encoding key regulators, which are frequently reused during different stages of development (Kleinjan and van Heyningen, 2005) and which have complex expression profiles. Generally, it is difficult to directly identify the disease-causing gene because the chromosomal disruption might lie in one gene but affect the expression of a nearby disease-causing gene as well. Apart from epigenetics and chromosomal rearrangements, mutations in transcription factors or in the regulatory DNA sequences influence gene expression profiles (Villard, 2004). For a gene to be expressed, one of the first steps is the formation of the transcription initiation complex at the minimal promoter region. This initiation complex consists of RNA polymerase II and other DNAbinding proteins (so-called basal transcription factors). At least 40 basal transcription factors have been identified to assemble with RNA polymerase II to start transcription. Numerous additional proteins are known to bind to other regulatory DNA sequences influencing gene transcription by, for example, recruitment of transcription factors or chromatin remodeling proteins. Mutations in transcription factors or in regulatory sequences can result in disproportional gene expression and cellular dysfunction, as documented for Hemophilia B Leyden. Generally, Hemophilia B is caused by mutations in the region coding for blood clotting factor IX. In a small number of cases, however, the mutation is located in the regulatory sequence of this gene (within 20 base pairs of the

134

Visser et al.

transcription initiation site), resulting in the prevention of binding of essential transcription factors. The absence of binding of certain transcription factors, leading to a lack of expression of this gene, can be compensated for during puberty by binding of the androgen receptor in the same promoter region (Crossley et al., 1992). Similarly, several diseases have been associated with a mutation in a transcription factor—mutations in the gene coding methyl-CpG– binding protein 2 (MeCP2) are known to result in Rett syndrome, a severe neurodevelopmental disorder (Gabellini et al., 2004). Even alterations of general transcription factors have been identified, for instance TFIIH mutations that result in a Cockayne dwarfism syndrome or in trichothiodystophy (Villard, 2004). In this chapter, we will first describe different approaches to treat diseases caused by dysregulated gene expression. Special attention will be given to the novel technology of engineered transcription factors. In the second part, the influence of epigenetic parameters on the function of such engineered transcription factors will be discussed.

II. TRANSCRIPTIONAL THERAPY As described in an earlier section and in the accompanying chapter (Verschure et al., Chapter 5, this issue), the knowledge about the functional organization of the human genome and on diseases, which are associated with a disturbed gene expression levels is accumulating. Several new technologies drawing on such knowledge have been introduced into the clinic, including: (1) agents that influence epigenetic gene control systems, (2) small interfering RNAs to downregulate gene expression, and (3) the introduction of new genetic material, that is, gene therapy. Gene therapy represents the most advanced experimental therapy in this respect. Alternatively, (4) engineered transcription factors can now be designed to interfere with gene-expression levels by either up- or downregulation of a specific gene. The clinical relevance of these different approaches will be discussed in a later section.

A. Epigenetic therapy Gene activity depends to a large extent on epigenetic factors (see also accompanying chapter, Verschure et al., Chapter 5, this issue). Agents have been developed to interfere with such epigenetic gene control systems. Several agents that alter DNA methylation patterns on DNA (e.g., Zebularine) or modifications of histones are being tested in the clinic, mainly to treat cancer (Egger et al., 2004). In addition, reexpression of the fetal globin gene to compensate for the lack of adult globin is giving some promising results in the treatment of

4. Successes and Pitfalls of Engineered Transcription Factors

135

hemoglobinopathies (Saunthararajah et al., 2003). Unfortunately, such agents are nonspecific and can show cytotoxicity, especially at high doses. Antisense oligonucleotides have been designed to bind and inhibit specific DNA methyl transferases to improve safety profiles. Also, for inhibitors of histone deacetylases (HDACs) that are used in clinical trials, all three classes of HDACs are inhibited. Clearly, more precise targeting agents are required. In fact, combinations of the above mentioned approaches are promising to increase efficiency of epigenetic therapy while lowering toxicity (Egger et al., 2004).

B. Posttranscriptional gene silencing Interference with protein production by manipulating the expressed RNA molecules has been shown useful in diverse areas of biological research, including reverse genetics and pharmaceutical research. Antisense oligodeoxyribonucleic acids (ODNs), ribozymes, and RNA interference (Dorsett and Tuschl, 2004) are possible approaches to interfere with RNA expression. Antisense molecules decrease RNA expression in two ways—either the RNA molecule in the formed DNA–RNA hybrid is degraded by RNase H, or binding of the ODN to the RNA molecule will inhibit translation or splicing by steric hindrance. So far, one antisense ODN has been approved by the FDA for clinical administration to treat cytomegalovirus infections of the eye (Vitravene by Isis, Inc). Alternatively, ribozymes, RNA-based enzymes, which can degrade RNA molecules by hydrolyzing the phosphodiester backbone, can be designed and applied. Several ribozymes have been studied in clinical trials, but none has reached the status of clinical approval. Most studies suggest so far that small interfering RNA (siRNA) molecules are by far the most potent factor for gene silencing (Dorsett and Tuschl, 2004). SiRNA acts by transferring so-called RNA induced silencing complexes (RISCs) to the target mRNA, which will subsequently be degraded. In addition, evidence is accumulating that siRNA molecules can silence gene expression directly at the DNA level (Kawasaki and Taira, 2004; Morris et al., 2004). Two siRNA molecules are in clinical trials, both against vascular endothelial growth factor A (VEGF-A) to treat age-related macular degeneration, the leading cause of adult blindness in the developed world. These reagents are either directly injected into the eye (Cand5, Acuity Pharmaceuticals, Inc) or genetically incorporated in adenoassociated virus (by Sirna Therapeutics, Inc). Several other siRNA clinical trials are planned, including one for Huntingtons disease (Sirna Therapeutics, Inc). As for all therapies, the major issues of these experimental therapies are delivery efficiency and specificity. For siRNA, research on delivery options resulted in promising in vivo methods—siRNA molecules can be complexed with cholesterol for efficient uptake into the liver and jejunum after intravenous

136

Visser et al.

administration (Soutschek et al., 2004). Alternatively, gene therapeutic carriers (plasmid-based (Wannenes et al., 2005), liposomal (Urban-Klein et al., 2005), and viral (Xia et al., 2004) have been successfully used for systemic, intratumoral, or intracellebellar delivery, respectively. The advantage of the latter two is that these approaches can be combined with targeting strategies to increase cell specificity of the therapy. Regarding target RNA specificity, several studies reported gene-specific siRNA-induced knockdown using microarray screens (Agrawal et al., 2003). However, it seems that the target specificity is not always perfect (Couzin, 2004; Jackson et al., 2003; Scacheri et al., 2004). Out of 359 published siRNA sequences, 75% could have aspecific effects (Snove, Jr. and Holen, 2004). However, using other hardware these authors could identify numerous unique siRNAs per target, suggesting that off-target effects are unnecessary. In this respect, computational analyses predict the lowest off-target destruction rate for RNA molecules of 21 nucleotides, which is the size of siRNA mainly found in nature (Qiu et al., 2005). In addition, administration of siRNA has resulted in side effects, like a pronounced interferon response that interfere with the therapeutic effects (Sledz et al., 2003). Despite these drawbacks, expectations of siRNA molecules as therapeutics are high, and much effort is being devoted to optimize this approach (Leung and Whittaker, 2005).

C. Gene therapy Gene therapy, the introduction of genetic material to achieve a therapeutic effect, was originally viewed as a promising approach to treat monogenetic diseases by compensating for the disease-causing mutation. The first clinical gene therapy trials started in 1990 for severe combined immunodeficiency disease (SCID), by introducing the normal copy of the adenosine deaminase gene into T lymphocytes by retroviruses (Blaese et al., 1995). In 1993, a trial was started for cystic fibrosis in which adenoviral introduction of the normal copy of the cystic fibrosis transmembrane conductance regulator gene was investigated (Korst et al., 1995). To date, more than 1000 clinical trials have been initiated enrolling thousands of patients. The majority of these (mainly phases I and II) trials, however, do not address monogenetic diseases but attempt to find effective treatment strategies for cancer (66%) (see www.wiley.uk.co/genetherapy/ clinical for more information). The fact that monogenetic diseases take up only 9% of all gene therapy trials represents one of the main advantages of gene therapy technology—not only healthy copies of naturally occurring human genes are suitable for introduction, also nonhuman genes and improvements to genes, such as addition of a secretion signal, can be achieved. Also for diseases, which are associated with insufficient expression of otherwise intact versions of genes, gene therapy holds great promise. It is interesting to note that another 9% of all clinical gene therapy trials is performed to increase blood

4. Successes and Pitfalls of Engineered Transcription Factors

137

vessel growth in order to treat vascular diseases. In these trials, the insufficient expression of growth factors is alleviated by transferring cDNA encoding (isoforms of) growth factors to the ischemic tissue. So far, however, these trails have been suboptimal, probably because combinations of (isoforms of) growth factors are required for mature vessel growth (Yla-Herttuala et al., 2004). Another advantage of gene therapy is that the transgene can be stably integrated in the human genome to permanently restore cellular functions. Unfortunately, integrating vector systems will cause random insertion of the DNA into the genome potentially causing harmful alterations in the host genome. This has been observed in otherwise successful SCID trials where the immunodeficiency has been corrected by gene therapy in 17 out of 18 patients (Cavazzana-Calvo and Fischer, 2004). Unfortunately, leukemia has been diagnosed in three patients, which seemed to be associated with the location of integration of the viral genetic material in the host genome (Hacein-Bey-Abina et al., 2003). Apart from this serious set back, integrated DNA might also suffer from promoter methylation, leading to insufficient expression levels. In general, inefficient and aspecific delivery of the transgene remains the major obstacle for gene therapy, and much research is focusing on developing specific and efficient gene transfer both through viral vectors (Verma and Weitzman, 2005) as well as nonviral delivery systems (Glover et al., 2005). Tremendous progress has been reported in increasing specificity by transductional (Everts and Curiel, 2004) or transcriptional (Sadeghi and Hitt, 2005) retargeting of the vectors. To increase efficiency of cancer gene therapy in particular, conditionally replication competent viruses have been constructed which in combination with chemotherapy resulted in some complete responses of head and neck tumors after intratumoral administration (Lin and Nemunaitis, 2004). Unfortunately, no follow-up phase III trials have been initiated for these successful trials. So far, only one gene therapy agent worldwide has reached approval for commercial clinical use and this concerns an adenovirus encoding p53 to treat head and neck squamous cell carcinoma in China (Peng, 2005).

D. Sequence-specific DNA-binding proteins as engineered transcription factors The above-described approaches are able to either downregulate endogenous genes by binding the relevant RNA molecules or to induce gene expression by introducing new DNA molecules. These approaches are based on known coding sequences. However, as the sequence of the total human genome has been elucidated, not only the coding sequence of genes are known, but also many of the regulatory sequences have been referred or will soon be identified. Such information can be exploited to design molecules that directly bind endogenous DNA sequences. These gene-specific DNA-binding molecules can be used for a

138

Visser et al.

broad spectrum of applications by targeting various effector domains to the DNA sequence of interest. It was shown that mutations can be repaired using site-directed cleavage of target DNA to increase the efficiency of homologous recombination for gene correction studies (Urnov et al., 2005). This new approach holds great promise for curing monogenetic diseases. These same DNA-binding modules can also be turned into engineered transcription factors by fusing transcription-activation or -repression domains to the DNA-binding domains (Gommans et al., Chapter 5, this issue). This way, the same DNAbinding molecule can theoretically be used to either up- or downregulate gene expression. In the accompanying chapter of this issue, the modes of action of such activation and repression domains are described in more detail (Verschure et al., Chapter 5, this issue). The engineering of transcription factors opens new possibilities to interfere with normal cellular functions in a very versatile manner. Of therapeutic relevance is the fact that such engineered transcription factors can potentially normalize disrupted gene-expression levels. Several advantages exist over other methods (summarized in Table 4.1), which make this technology a powerful and promising feature of biomedical research. The main advantage of inducing endogenous gene expression over introducing the cDNA encoding a particular gene is that engineered transcription factors will induce expression of all isoforms of a particular gene in the correct stoichiometric ratios. As gene therapy vectors are limited by insert size, only one isoform of a gene can generally be introduced. Yet, one transcription factor can be constructed to induce or repress a whole gene family simultaneously by targeting a common DNA sequence. Other advantages of engineered transcription factors, both activating and repressing, include their small size. This allows simultaneous delivery of several different transcription factors providing new approaches to treat multifactorial diseases. In addition, genetic manipulation of engineered transcription factors makes it possible to introduce cellular uptake sequences, which will yield in a high penetration of the TF in the targeted tissue. Moreover, several delivery options are available for TF—the transcription modulators can be administered as nucleic acids, as proteins (fused to e.g., protein transduction domains) (Tachikawa et al., 2004) or even as small chemical molecules when formulated as polyamides (Melander et al., 2004). In downregulating gene expression, the main advantage of engineered transcription factors lies in the fact, that, in general, only two copies of a gene need to be targeted per cell instead of multiple RNA copies, as is the case in siRNA approaches. Using DNA-binding molecules permanent gene silencing might be achieved by targeting enzymes effecting histone modifications or DNA methylation to the genes to-be silenced (Table 4.1). Transcription factors minimally consist of a DNA-binding domain and a transcriptional activation or repression domain. Different approaches have

4. Successes and Pitfalls of Engineered Transcription Factors

139

Table 4.1. Characteristics of Engineered Transcription Factors Versus Other Experimental Therapies

ZF-TF Up- and downregulation All splice variants Formulations

Multigene modulation Specificity Permanent gene silencing Efficiency

Gene therapy

siRNA

HDAC inhibitors/DNA demethylation agents

Yes

No

No

No

Yes

Not easily

Possibly

Yes

Nucleic acid, protein, chemicals Yes

Nucleic acid

Nucleic acid

Chemicals

No

No

Gene specific Possibly

Delivery issues

Off target effects Possibly (methylation) Numerous RNA copies/cell

Yes, but aspecific Not specific

2 DNA mc/cell

Yes, but random integration Delivery issues

Yes

The different advantages of zinc-finger–based transcription factors are mentioned in column 1 and compared with other experimental therapeutic strategies for gene expression modulation including gene therapy, small interfering RNA or HDAC inhibitors.

been exploited to develop sequence specific DNA-binding molecules (Uil et al., 2003). The most advanced agents in this respect include chemical polyamides, triplex forming oligos (TFOs, synthetic DNA molecules) and engineered zincfinger proteins (ZFPs). All three approaches have the ability to modulate gene expression in living cells by binding to genes in the chromatin context (Uil et al., 2003). DNA-binding polyamides (Fig. 4.1) consist of pairs of hydroxypyrrole, imidazole, and pyrolle molecules forming antiparallel stretches. Specific combinations of these molecules recognize a particular DNA base pair. Although nuclear uptake seems to be quite inefficient for polyamides, its potential of interfering with gene-expression levels in living cells has been demonstrated. Polyamides as well as polyamides fused to effector domains have been constructed and have been shown to be able to interfere with endogenous geneexpression levels (Uil et al., 2003). Importantly, a polyamide fused to an alkylating agent was identified from a library to downregulate expression of the histone 4c gene; this resulted in tumor regression after intravenous administration (Dickinson et al., 2004). Although polyamides are believed mainly to limit the transcription of a gene by obscuring the binding site of a natural

140

Visser et al.

Figure 4.1. Hairpin polyamides bind in the minor groove of double-stranded DNA according to a framework in which combinations of pyrrole/imidazole, pyrrole/hydroxypyrrole, hydroxypyrrole/pyrrole, and imidazole/pyrrole bind to C-G, A-T, T-A, and G-C base pairs, respectively. Adapted from Uil et al. (2003), by permission of Oxford University Press.

Figure 4.2. Binding of triplex forming oligos to the major groove of double-stranded DNA. Binding involves Watson–Crick (W-C) hydrogen bounds and either Hoogsteen (H) hydrogen bounds for the parallel binding to pyrimidine-rich stretches of DNA or reverse Hoogsteen (RH) hydrogen bounds for antiparallel binding. For the parallel binding, protonation (þ) of the cytosine is required, which prevents the occurrence of this type of binding in physiological conditions. Adapted from Uil et al. (2003), by permission of Oxford University Press.

transcription factor (Gearhart et al., 2005), many aspects of their mode of action are still to be clarified. Triplex forming oligos (Fig. 4.2) are single-stranded DNA molecules that bind in the major groove of DNA to a purine-rich target strand, thereby forming a triple helical structure. Because of their potency to induce recombination, TFOs (with or without fused DNA damaging agents) are potentially powerful agents applicable gene correction studies. Alternatively, TFOs have been shown to downregulate endogenous gene expression, either by interference with transcription initiation through competition with endogenous transcription

4. Successes and Pitfalls of Engineered Transcription Factors

141

Figure 4.3. (A) Structure of the major groove binding zinc-finger proteins. Zinc-finger proteins consist of highly conserved modules (“fingers”), which are made up by two sheets and an helix, held together via a zinc ion (Zn) coordinated by two cysteines (C) and 2 histidines (H). The amino acids primarily involved in DNA recognition are represented by the boxed position in the helix. Adapted from Uil et al. (2003), by permission of Oxford University Press. (B) Crystal structure of two copies of the threefinger Zif268 protein (dark blue) bound to adjacent sites on DNA (light pink). Zinc atoms are indicated as light blue spheres (PDB ID code IP47; Peisach and Pabo, 2003). The six zinc-fingers recognize the DNA sequence specifically in the major groove and circle the DNA about 1.5–2 times. (See Color Insert.)

factors, or by prevention of transcription by inhibiting transcription-initiation complex formation (Uil et al., 2003). The therapeutic potential of such agents has been shown by a TFO directed against type alpha1(I) collagen, which resulted in a decrease in collagen production after intravenous injection in fibrotic rats (Cheng et al., 2005). In addition, oligos have been exploited as DNA-binding domains in engineered transcription factors; but this has, to the best of our knowledge, not been exploited yet for endogenous modulation of gene expression. Critical issues in TFO research for clinical use include the improvement of binding affinity and selectivity, as well as stability. Regarding the latter, expression vectors for single-stranded DNA are available to administer TFOs as gene therapeutics, increasing their delivery efficiency (Datta and Glazer, 2001). So far, the most flexible platform for engineering gene-specific DNAbinding molecules is the construction and stitching together of zinc-finger modules. Zinc-finger proteins (Fig. 4.3) are the most abundant transcription factors in eukaryotes, consisting of highly conserved modules of approximately

142

Visser et al.

30 amino acids. Every module (“finger”) can bind three to four base pairs, and a stretch of only seven amino acids is responsible for interaction with the target DNA (Gommans et al., Chapter 5, this issue). The stretch of seven amino acids containing the sites of DNA interaction can be easily manipulated to bind specific triplets of DNA (Segal, 2002). The stitching together of 6 fingers results in the binding of 18 base pairs, which can be computed to be a unique sequence in the human genome. This gene-specificity might represent an advantage over polyamides, as these chemical molecules are generally limited to bind maximally 10–11 base pairs. Several genes have been successfully targeted by engineered ZFPs fused to either repressor domains or activation domains, as described in detail in a later section. Several research groups and a biotech company (Sangamo Biosciences) have made major progress in engineering these zinc-finger–based TFs (ZF-TFs). A clinical trial has started to evaluate a plasmid-encoded ZF-TF to upregulate the expression of vascular endothelial growth factor A, for induction of angiogenesis in peripheral artery disease (Rebar, 2004). In a later section, we summarize the 14 genes (see Table 4.2) targeted for up- or downregulation by zinc-finger–based transcription factors.

1. Zinc-finger–based TFs induced downregulation of endogenous gene expression Zinc-finger–based TFs provide a new approach for altering disturbed gene-expression patterns to treat numerous diseases. Several genes have been targeted by these agents to investigate their relevance as therapeutics in cancer treatment. For example, overexpression of the erbB-2 gene is correlated with a poor prognosis in cancer, and downregulation would result in an inhibition of tumor cell proliferation. An engineered six-finger ZF-TF, targeting a DNA sequence in the 50 -untranslated region (50 -UTR) of the oncogene erbB-2, has been coupled to the repressor Kox-1 KRAB domain (Beerli et al., 1998, 2000a). After retroviral introduction of the sequence encoding this ZF-TF, an accumulation of infected cells in G1 phase was observed, showing the potential therapeutic applicability of downregulating erbB-2 expression in cancer therapy (Beerli et al., 2000a). Similarly, a six-finger protein recognizing the 50 -UTR of a related receptor tyrosine kinase family member erbB-3, was coupled to the KRAB repressor domain. Flow cytometry revealed that erbB-3 expression was abolished in the 74% of the infected cells. No effect of the erbB-2 ZFP was seen on erbB-3 gene expression and vice versa, although the 18 base pair target DNA only differs by 3 base pairs. Checkpoint kinase 2 (CHK2) is a cellular regulator of the p53 tumor suppressor gene’s activity and plays a role in cell-cycle progression, DNA damage, and cell death. Therefore, CHK2 is a possible therapeutic target to be

Table 4.2. Endogenous Genes Successfully Targeted for Modulation of Gene Expression by Engineered Zinc-Finger Transcription Factors Target gene

Target sequence determination

Number of fingers

ED

Reference(s)

erbB-2, erbB-3

Promoter region þ library selection

23

KRAB, VP64

CHK2

32

KRAB

MDR1

DNase hypersensitive area around transcription start site Library

2þ1þ2

KRAB, VP16

VEGF-A

DNase hypersensitive area

3

vErbA, TR?, HMT, VP16, p65

Oct-4

Close to transcription start site, selected from 10 prescreened ZFP DNase hypersensitive area Consensus binding sites Library Library TF binding sites Alu repeats (positioned nucleosome), micrococal nuclease sensitive 500 bp around promoter Downstream transcription initiation site

32

KRAB, VP16

(Bartsevich and Juliano, 2000; Xu et al., 2002) (Liu et al., 2001; Snowden et al., 2002, 2003) (Bartsevich et al., 2003)

6 3þ2 6 6 6 3

KRAB, VP16 VP16 VP64 VP64 KRAB, VP64 VP16

(Ren et al., 2002) (Falke et al., 2003), (Blancafort et al., 2003) (Magnenat et al., 2004) (Graslund et al., 2005) (Zhang et al., 2000)

3 3

VP16, p65, vErbA, TR VP16, p65, vErbA, TR

(Jouvenot et al., 2003) (Jouvenot et al., 2003)

PPAR Bax CDH5 ICAM-1

-Globin Epo IGF2 H19

(Beerli et al., 1998, 2000a; Lund et al., 2004) (Tan et al., 2003)

Approaches to identify the target sequences, the number of fingers, and the effector domains used to modulate the expression of the different genes targeted within the chromatin environment are presented.

144

Visser et al.

downregulated in cancer treatment. A six-finger protein, designed to bind the CHK2 promoter, has been coupled to the KRAB repression domain (Tan et al., 2003). This transcription factor was able to cause a 10-fold reduction in CHK2 mRNA levels in two different cell lines, which resulted in barely detectable protein levels and was associated with a loss of DNA damage-induced CHK2 dependent p53 phosphorylation. As modulation of CHK2 expression should not have any downstream effects in undamaged cells, CHK2 serves as a suitable model to demonstrate the strict gene specificity of engineered ZF-TFs in modulating gene expression. Expression of this ZF-TF did not influence the gene expression of 16,000 other genes in two different cell lines (Tan et al., 2003). This study demonstrates the remarkable gene-specific modulation using engineered six-finger proteins. The P-glycoprotein is another clinically relevant target for cancer therapeutics. This P-glycoprotein is capable of transporting anticancer drugs out of the cell, causing resistance to chemotherapy. To downregulate P-glycoprotein gene expression, a hybrid ZFP was constructed consisting of two zinc-finger domains of the human transcription factor Sp1, one zinc-finger domain of murine Zif268, and two newly synthesized domains (Bartsevich and Juliano, 2000). After fusing two KRAB-A domains to this ZFP, the ZF-TF inhibited endogenous expression of the P-glycoprotein 15-fold (Bartsevich and Juliano, 2000). Altered expression profiles of 3–6-fold differences were observed for only 7 out of 2059 other genes. In a subsequent study, the possible therapeutic effect of this constructed ZF-TF was evaluated (Xu et al., 2002). Here, a breast carcinoma cell line that highly overexpressed P-glycoprotein was stably transfected with DNA encoding the ZF-TF (described in an earlier section) under the control of an inducible promoter. When this five-finger protein was expressed, a significant reduction of over 90% in P-glycoprotein expression was seen. This reduction was associated with an increased rate of a P-glycoprotein substrate accumulation inside the cells. A substantial rise in cytotoxic effects of the antitumor drug doxorubicin in a dose-response profile was seen, causing a 10-fold increase in drug sensitivity after the introduction of the ZF-TF (Xu et al., 2002). Instead of killing tumor cells directly, the inhibition of new bloodvessel formation (angiogenesis) provides a potentially powerful approach to shut off the oxygen and nutrient supply to the tumor cells thereby inhibiting tumor cell growth. VEGF-A is an important factor in angiogenesis, and upregulation of VEGF-A has been associated with a poor prognosis in cancer patients. Downregulation of VEGF-A in tumor tissue therefore should cause a decrease in tumor vessel growth, which subsequently could result in tumor cell death. Therefore, ZF-TFs were constructed for downregulation of VEGF-A expression (Snowden et al., 2003). Each protein consisted of three zinc-finger DNA-binding domains coupled to the repressor domain vErbA. In a transient transfection study, each of

4. Successes and Pitfalls of Engineered Transcription Factors

145

these ZF-TFs inhibited VEGF-A MRNA expression down to 50%. To eliminate the influence of untransfected cells, a stable inducible cell line was constructed for one of the ZF-TFs. After induction, this expressed ZF-TF strongly repressed VEGF-A protein production. Also, in a stably transfected highly vascularizing tumorigenic cell line, a 20-fold reduction in VEGF-A protein expression was detected after induction of the ZF-TF. The reduction seen in this cell line brought the VEGF-A protein level back to the level observed in a nonangiogenic cell line (Snowden et al., 2003). This observation suggests that the observed repression would be efficient enough to suppress tumor angiogenesis. Alternatively, a more permanent silencing of gene expression might be achieved by coupling DNA-binding domains to enzymes, which can directly interfere with epigenetic gene control systems. To this end, experiments were conducted with the same zinc-fingers as described in an earlier section, coupled to the minimal catalytic domain of the enzyme histone methyl transferase (Snowden et al., 2002). Repression of endogenous VEGF-A expression was accomplished through local methylation of histone H3 and was comparable to repression accomplished by the v-ErbA domain (Snowden et al., 2002). This report therefore presents an alternative approach to repress gene expression, in which transient expression of the engineered ZF-TF might result in a long-term repression of gene transcription. In addition to the above cancer related targets, other types of genes have been targeted by zinc-finger–based TFs. One six-finger protein linked to a KRAB repression domain has been engineered to control the fate of stem cells by repressing a gene known to play a role in stem cell differentiation (Oct-4) (Bartsevich et al., 2003). This ZF-TF inhibited expression of Oct-4 up to threefold, thereby altering the morphology of the stem cells. Another study involving regulation of an endogenous gene (peroxisome proliferator-activated receptor [PPAR] ; Ren et al., 2002) illustrates the power of engineered transcription factors in fundamental research. This PPAR gene consists of two splice variants 1, and 2, and plays a role in adipogenesis (formation of fat cells). Both isoforms share the same C-terminus expressed from the same promoter, while the translated amino terminus of splice variant 2 is regulated from a different promoter. To delve into regulation of these factors, two 6-finger ZFPs were coupled to the repression domain KRAB. One ZF-TF was capable of almost complete inhibition of the induced upregulation of both splice variants, while the other zinc-finger selectively lowered splice-variant 2 levels to approximately 50% of normal. The first ZFP was subsequently used to knock down the expression of PPAR , after the two splice variants were reintroduced separately by retroviral infection to determine the role of the individual variants in adipogenesis (Ren et al., 2002). These studies indicate the broad applicability of ZF-TFs in many different cell types.

146

Visser et al.

2. Upregulation of endogenous gene expression by engineered ZFPs In addition to downregulation of gene expression as a novel approach in cancer therapy, upregulation of specific genes might also result in inhibition of tumor cell growth. Among these candidates are target genes of the transcription factor p53, which is frequently mutated in cancer cells. One of the many targets of p53 is Bax, which induces the release of cytochrome c from the mitochondria, leading to caspase-dependent apoptosis. A ZF-TF was constructed to upregulate the expression of Bax, which indirectly induces apoptosis when p53 is no longer functional (Falke et al., 2003). This engineered ZF-TF consisted of five modular zinc-finger domains (three domains from the naturally occurring transcription factor Zif268 and two newly synthesized domains), attached to the minimal activation domain derived from the herpes simplex viral VP16. After transiently transfecting cells with a plasmid encoding the designed ZF-TF, a moderate upregulation in Bax—but not in p21 expression, another p53 target gene—was observed and resulted in reduced cell viability (Falke et al., 2003). As described in an earlier section, downregulation of VEGF-A represents a powerful anticancer strategy that involves inhibiting of new blood vessel formation. Similarly, induced upregulation of VEGF-A represents a powerful approach to induce angiogenesis. Therapeutic angiogenesis is warranted in several pathophysiological conditions, including ischemic organ failure and heart diseases. Phase II clinical trials are being performed with (adenoviral) delivery of a gene encoding one VEGF-A isoform to induce vessel growth. These studies however, had limited success, as the induced vessels are immature and leaky (Rajagopalan et al., 2003). The general consensus that, in order to induce functional mature vessels, all naturally occurring isoforms of different growth factors would be required. Engineering transcription factors provide a powerful strategy in this respect, as the expression of all isoforms is induced, and the expression of several growth factors can be induced simultaneously. To induce angiogenesis by upregulation of endogenous VEGF-A expression, three-finger proteins were engineered and coupled to the activation domain VP16 or p65 (Liu et al., 2001). These ZF-TFs increased VEGF-A levels up to 15-fold, which exceeded levels normally induced by hypoxia. Importantly, all the functionally different splice variants of the VEGF-A gene (Carmeliet et al., 1999) proportionally increased after ZF-TF-induced upregulation of VEGF-A expression (Liu et al., 2001). The genes encoding ZFPs fused to VP16 activation domains were introduced in recombinant adenoviral vectors, which were injected in mice and improved experimental wound healing and formation of new mature blood vessels was observed (Rebar et al., 2002). The neovascularization after activation of endogenous VEGF-A by the zinc-finger

4. Successes and Pitfalls of Engineered Transcription Factors

147

constructs was compared with the neovascularization after introduction of VEGF-A164 cDNA. Although the mice ears treated with an adenovirus expressing VEGF-A164 showed increased vessel growth, the vessels were hyperpermeable as detected by Evans blue dye infusion. On the contrary, vasculature in the mice ears treated with an adenovirus containing the zinc-finger construct was not permeable for Evans blue dye. This suggests that modulating endogenous VEGF-A expression induces the formation of more mature vessels, pointing to the importance of inducing all splice variants (Rebar et al., 2002). Also, in a clinically relevant rabbit hind limb ischemia model, capillary density and blood flow increased about twofold in animals treated with VEGF-A ZF-TF after intramuscular injection of a plasmid encoding ZF-TF, compared with the effects of irrelevant plasmids (Dai et al., 2004). Based on these in vivo data, a clinical trial has started to investigate an engineered ZF-TF for the induction of VEGF-A in cardiovascular disease (Rebar, 2004). Other genes playing a role in angiogenesis have also been targeted with engineered ZF-TFs, such as the vascular endothelial cadherin protein (CDH5), which plays a role in permeability of the endothelium. CDH5 was upregulated by a three-finger protein in all five different cell lines tested (Blancafort et al., 2003). Similarly, expression of the intercellular adhesion molecule (ICAM-1) was altered. Six-finger proteins were identified by screenings of phage-display libraries (Magnenat et al., 2004). After further optimization of DNA-binding specificity by protein engineering, one ZFP coupled to VP64, increased ICAM-1 expression up to 135-fold, exceeding the degree of inducing ICAM-1 expression by naturally occurring cytokines. The alterations in ICAM-1 expression were specific for ICAM-1, as the expression of seven other endothelial cell markers, determined in two different cell lines, were not affected by this ZF-TF (Magnenat et al., 2004). Other endogenous genes targeted for upregulation are erythropoietin (Epo) and -globin. Erythropoietin controls the biogenesis of erythrocytes and has been upregulated by a three-finger protein coupled to VP16, resulting in an increase in Epo from 0 mU/ml up to 200 mU/ml (Zhang et al., 2000). Induction of the normally silenced fetal -globin can alleviate the symptoms of sickle cell disease, which arises from a mutation in the -globin gene. Six zinc-finger domains attached to the activation domain VP64 induced fetal -globin expression up to 14-fold and could therefore compensate for the mutation in the -globin gene (Graslund et al., 2005). This ZF-TF presents a new and promising approach for the treatment of sickle cell disease. ZF-TFs are also capable of inducing the expression of otherwise stably silenced genes. ZF-TF induced upregulation of paternal-repressed H19 gene or the maternal-repressed IGF2 gene in mouse–human cell hybrids containing one human chromosome of either paternal or maternal origin (Jouvenot et al., 2003).

148

Visser et al.

III. ENGINEERED ZINC-FINGER–BASED TRANSCRIPTION FACTORS (ZF-TFs) AND THE INFLUENCE OF NUCLEOSOMES The number of genes successfully targeted by ZF-TFs demonstrates the high potential of these engineered factors for modulating gene activity. We will address some challenges and possible solutions for successful molecular design by taking a closer look at the effect of ZF-TFs at the level of in vivo DNA/ chromatin structure. In simple terms, a successful ZF-TF needs to fulfill two criteria: (i) the DNA-binding module needs to bind specifically to the selected DNA in the chromatin environment in vivo and (ii) the effector-domain needs affect gene expression efficiently. This second point is addressed in detail in the accompanying chapter (Verschure et al., Chapter 5, this issue). A set of rules to help the design of engineered ZF-TFs to successfully bind DNA in vivo can be distilled from the pilot work reviewed in this chapter. The engineering of zincfinger modules that bind naked DNA at a specific sequence is well formulated (this is in particular due to the efforts of Sangamo and several research groups that defined proprietary design aspects, which will not be discussed here (Liu et al., 2002; Magnenat et al., 2004; Segal et al., 2003; Sera and Uranga, 2002). Even so, the effectiveness of ZF-TFs in the cellular environment still mostly depends on well-guessed trial and error. To a large extent this is caused by the fact that DNA is not “naked” in vivo but is packed with proteins and organized into chromatin.

A. ZFP binding and nucleosomal structure In vivo, DNA is organized by histones into chromatin, which is believed to limit ZF-TF binding. The core histones (H2A, H2B, H3, and H4) form nucleosomes, which each wrap about 150 bp of DNA in 1.7 turns along their surface (Luger et al., 1997). On average, nucleosomes are spaced every 200 bp, leaving roughly a 55-bp linker DNA between two nucleosomes. The nucleosomal arrays (beadson-a-string) are organized into 30-nm wide chromatin fibers and subsequently into higher order structures (see accompanying chapter for more details (Verschure et al., Chapter 5, this issue). The wrapping of the DNA around the nucleosome limits the accessibility of the DNA for regulatory proteins such as ZF-TFs (Collingwood et al., 1999; Urnov, 2002, 2003). Crystal structures of nucleosomal particles predict that transcription factors can only bind six to eight consecutive base pairs before being sterically blocked by histones and by fluctuations in the (minor and major) groove width (Edayathumangalam et al., 2004). The crystal structure of a three-zinc-finger module bound to naked DNA shows that the binding of the ZFP follows the major groove (Pavletich and Pabo, 1991). As one turn of the DNA helix consists of 10 base pairs, larger zinc-finger modules engineered to recognize 18 bp therefore wrap around the DNA about

4. Successes and Pitfalls of Engineered Transcription Factors

149

Figure 4.4. Crystal structure of nucleosome with DNA (PBD ID code 1AOI; Luger et al., 1997) viewed from two different angels (A and B) and shown in sphere representation. DNA (dark and middle gray) is wrapped around the histone complex (light gray). The close contact of the DNA with the histones (arrow head) indicates that ZF-TF cannot follow the major groove for a full helical turn (10 bp) in an undisrupted nucleosome. (C) It is hypothesized that by creating ZF-TF with a linker that can span several base pairs of unbound DNA, binding to accessible regions of nucleosomal DNA may be facilitated (connected arrows). (D) The alignment of the DNA helix around the nucleosome forms “supergrooves” that can be physically linked (black line) by targeting two sequences spaced by about 80 bp (corresponding to one turn around the nucleosome) (PBD ID code 1S32; Edayathumangalam et al., 2004).

1.5–2 times (Peisach and Pabo, 2003) (see Fig. 4.3 B). A crystal structure of a natural 5-finger module indicates that the first finger does not bind the naked DNA, while fingers 2–5 binding 12 base pairs do circle the DNA (Pavletich and Pabo, 1993). It is therefore believed that such large ZF-TFs cannot bind DNA that is wrapped around nucleosomes (see Fig. 4.4). If ZF-TF bind nucleosomal DNA, it is believed that a confirmation change is required by shifting the nucleosome to one side or by creating a DNA loop (Chakravarthy et al., 2005). It is more likely, however, that the larger ZF-TF will only bind in

150

Visser et al.

nucleosome-free (linker) DNA. It is still not understood how ZFPs bind their DNA sequences in the chromatin environment in vivo. In vitro studies show that nucleosomal DNA can become accessible for DNA-binding proteins, such as LexA by spontaneous rapid conformational changes (Li and Widom, 2004; Li et al., 2005). Also, polyamides were shown to bind their recognitions sites with high affinity when these are only partially exposed at the nucleosomal surface (Gottesfeld et al., 2001). The linker regions between the individual fingers of ZFPs in solution are found to be highly flexible, which is believed to allow the protein to search along the DNA for the correct base sequence because the nonspecific association is weak. Once the correct base sequence is encountered, base-specific contacts are initiated in the major groove, thereby locking the zinc-finger on the DNA via a “snap-lock” mechanism (Dyson and Wright, 2004). Speculatively, such a mechanism may facilitate binding of the zinc-finger to nucleosomal DNA by initially binding a small, but exposed, DNA region and then snapping closed after a conformational change in the nucleosome structure has occurred that make the DNA better available. Most designed ZF-TFs are based on the structure of the human transcription factor SP1. SP1 consists of three fingers containing the DNA recognizing alpha helices, separated by short (conserved) linkers and targets an uninterrupted DNA sequence. In plants, however, most natural DNA-binding zinc-finger proteins have an entirely different structure in which individual zincfingers are typically separated by stretches of 19–232 amino acids (Takatsuji, 1998). These longer linker sequences allow a large spacing between different triplets of DNA recognized by a single ZFP-finger. These linkers can span more than 10 bp, which corresponds to one helical turn of the DNA (Kubo et al., 1998; Takatsuji and Matsumoto, 1996). Such spacing would allow a zinc-finger protein to bind to nucleosomal DNA when the two sets of recognition sequences are facing outward on different helical turns (see Fig. 4.4). In human, the transcription regulatory glucocorticoid receptor (GR) serves as an example for this kind of ZF-TF–DNA interaction. The GR-dimer binds two 6 bp sequences spaced by one helical turn of the DNA and can bind nucleosomal DNA (Collingwood et al., 1999). By adapting the linker design (Moore et al., 2001), a ZF-TF may be designed to bind such nonconsecutive sequences and facilitate binding of nucleosome associated DNA. Another approach to bind a specific DNA sequence, which is associated with a nucleosome has been reported (Edayathumangalam et al., 2004). Analysis of the crystal structure of the nucleosome showed that the two rounds of DNA that wrap the nucleosome align and together form a “supergroove” (see Fig. 4.4D). In such a supergroove, 14–16 bp can be targeted in a sequencespecific manner with an approximate 80-bp gap between the two target sets of 7–8 bp (80 bp corresponds to one circle around the nucleosome). DNA in such a

4. Successes and Pitfalls of Engineered Transcription Factors

151

supergroove has been successfully targeted by a designed polyamide. The crystal structure confirmed that this polyamide is bound in the expected supergroove (Edayathumangalam et al., 2004) (Fig. 4.4D). Each nucleosome has seven minor and six major DNA supergrooves that connect the two turns of the DNA around the nucleosome. Similar to minor-groove binding polyamides, major-groove binding ZFPs can thus be designed that specifically bind and connect two stretches of DNA sequences spaced by about 80 bp in nucleosomal context. Edayathumangalam et al. (2004) showed, however, that by linking the two gyres of DNA, dissociation of the nucleosome was inhibited, indicating that the DNA cannot unwrap from the nucleosome anymore. This flexibility is essential for processes such as DNA replication and transcription. Before a ZF-TF can be designed to link the two gyres of the DNA for therapeutics, the effect of ZF-TF association and dissociation frequencies on DNA replication and transcription should be studied in cell cultures.

B. Target definition In yeast, a genome-scale assay indicated that most occupied transcription-factor binding motifs are devoid of nucleosomes. This strongly suggests that nucleosome positioning is a determinant for a locus’ access to such factors (Yuan et al., 2005). For higher eukaryotic cells no genome wide information is available about positioned nucleosomes, although attempts are made to create a nucleosome positioning database (Levitsky et al., 2005). In general, it is believed that only few nucleosomes consistently occupy a precise position relative to the promoter DNA sequence. However, preferred positions may be acquired during gene activation (Georgel, 2005). Without the availability of nucleosome-position data, two approaches are taken to select ZFPs that should be able to bind chromatin (see Table 4.2). First, large phage-display libraries are screened, allowing nature to select for the most effective ZF-TF. For this approach, a large library is created containing three or six finger ZFP-modules fused to an activator domain. The library is introduced into a cell line by viral infection, and those cells in which the selected gene is expressed higher are selected by FACS sorting. From these cells, the ZFP-coding regions are obtained by PCR and recloned for further rounds of selection (Blancafort et al., 2003; Magnenat et al., 2004). This approach depends on the availability of a phage-display library and an efficient selection method (e.g., FACS analysis of immunostained cell surface receptors (Blancafort et al., 2003; Magnenat et al., 2004). The other approach involves selecting potential accessible target sites, based on putative transcription-factor binding sites and/or biochemical experiments, such as DNase sensitivity, followed by designing ZFPs to these sequences (see Table 4.2). In most studies, several ZF-TFs were initially engineered for a given gene, each targeting a different DNA sequence within the promoter region or

152

Visser et al.

just downstream of the transcription-initiation site. Usually only one or two of a series of ZF-TFs were able to modify endogenous gene activity. For instance, Zhang et al. (2000) engineered a set of 10 different ZFPs directed to 4 distinct sites near the EPO-gene initiation site. Two of these 10 ZFPs could activate the endogenous gene efficiently; both directed to the same DNA sequence (see Table 4.2). Two reports of these investigators (Liu et al., 2001; Zhang et al., 2000) illustrate computational, comparative, and biochemical parameters considered in the design of chromatin-binding ZF-TFs. These considerations are discussed in a later section.

1. Target sequence selection Digestion of chromatin by DNase I results in the identification of “DNase I hypersenstitive sites,” which are generally considered to be “open” chromatin structures and relatively more accessible to transcription factors. To help select target sequences, Liu et al. (2001) determined DNase I sensitivity of the promoter region of the VEGF-A gene. They also plotted the binding sites of the natural transcription factor SP1, assuming that natural TF have access to DNA. Furthermore, they evaluated sequence conservation among human, mouse, and rat, with the rationale that conserved regions may be involved in gene regulation and therefore accessible for transcription factors. There appeared to be no strict correlation between sequence conservation and DNase hypersensitivity. In order to evaluate the predictability of successful ZF-TF target selection in the context of chromatin, nine zinc-fingers were designed, targeted to three different DNase sensitive and two DNase insensitive sites. A reporter-plasmid assay, in which a luciferase reporter is driven by the VEGF promoter region, indicated that all of the ZFPs fused to the VP16 activating domain activated reporter-gene expression. A transiently transfected plasmid is, however, not expected to be fully chromatinized. Only the ZF-TFs targeted to the DNase hypersensitive sites were able to activate endogenous VEGF expression. As one of these nonhypersensitive sites was located in the hypoxia responsive element (which is DNase sensitive in various other cell lines), and the other in a conserved region, DNase hypersensitivity was originally believed to be the preferred parameter to determine target sequences. In contrast to the findings just described, Zhang et al. (2000) found that their most successful ZF-TFs targeted to a site that was a priori not DNase hypersensitive. Using DNase sensitivity, micrococcal enzyme digestion, and chromatin immunoprecipitation (ChIP) assays the ability of ZF-TF to bind chromatin was evaluated. No DNase I hypersensitive “hotspot” was found at the targeted site (in this case, 862 bp upstream of the transcription start site), or anywhere else in the promoter region. As this gene is normally silent in the cell type used, this was anticipated. Binding of the ZF-TF changed the chromatin

4. Successes and Pitfalls of Engineered Transcription Factors

153

structure and caused a DNase hypersensitive site to appear. Using ChIP assays, the authors showed that the ZF-TF did bind within the expected region. Importantly, the ZF-TF was found not to bind the same (9 bp) recognition sequence, which is present a few thousand kb away. This shows that DNA sequence quality is not the only binding determinant—chromatin structure appears capable of preventing the binding of a ZF-TF. To better understand how their ZF-TF got access to the DNA, they used a micrococcal nuclease digestion and computational sequence analysis. Micrococcal nuclease is considerably smaller than DNase I and gains efficient access to the linker DNA between nucleosomes (Drew, 1984). Sequence analysis showed that the –862 site is located at the 50 end of an Alu element. Alu’s are repetitive DNA elements that are known to position nucleosomes (Englander and Howard, 1995). The authors conclude that the ZF-TF probably binds to nucleosome-free linker DNA that is established by the positioning of the nucleosome by the neighboring ALU repeat. Only a few studies have assessed the influence of nucleosome position on ZF-TF binding. The relevant results suggest that designing ZF-TFs to bind in particular to nucleosomal free (linker) DNA enhances the chance of effectiveness. Similarly, DNase I sensitivity might be an efficient approach to determine potential target sites, although several reported sites were not a priori DNase I hypersensitive, but were effectively targeted by ZF-TFs (Lund et al., 2004; Van Eenennaam et al., 2004; Zhang et al., 2000). Knowledge about the positioning of nucleosomes may help guide target sequence selection by choosing sequences expected to act as nucleosome-free linker DNA. It is therefore promising that documentation of nucleosomal positions has begun to be put into database (Levitsky et al., 2005). Also, the approach designed to systematically study the position of nucleosomes in yeast (Yuan et al., 2005), may be applicable for higher eukaryotes as well. However, this approach depends on the availability of highresolution (tiling) microarrays, which at present must be custom made and can only cover part of the human genome. In absence of experimental data, another good alternative to select ZF-TF target sites, is by selecting sites close to binding sites of natural regulation proteins (Graslund et al., 2005). The validity of this approach is indirectly supported by work of Lund et al. (2004). These authors used a phage-display library to select 13 ZF that bind in vitro naked DNA representing the 1.4-kb promoter region of the ErbB-2 gene. These ZF were subsequently fused to a VP64 activation domain or a KRAB repression domain. The ability of these ZF-TF to, respectively, induce or repress transcription of the ErbB-2 gene was analyzed. In addition, the ZF-TF target sites were plotted along the promoter region together with DNase hypersensitive sites and binding sites of known transcription factors. In Fig. 4.5 the data of Lund et al. (2004) is summarized. The comparison between ZF-TF induced transcriptional activity

154

Visser et al.

Figure 4.5. Summary of ZF-TFs targeted to the ErbB-2 promoter region; data taken from Lund et al. (2004) and Beerli et al. (2000a). The cartoon indicates the spatial position of the ZF-TF binding sites (arrows) with respect to the transcriptional start site (hooked arrow), DNase I sensitive area (Blue box) and natural transcription factor binding sites (black ovals). In the table below are summarized per ZF-TF the position in base pairs upstream of the transcription start site, the affinity for naked DNA (in nM), the ability to upregulate transcription when fused to VP64 and the ability to downregulate the gene when fused to KRAB. Note that some of the three-finger ZF-TF bind to several sites. *The ZF-TF16 has six fingers. No absolute rules can yet be distilled to predict ZF-TF activity, although it is clear that the position where the ZF-TF is targeted to is important for its efficiency. (See Color Insert.)

versus chromatin characteristics of the targeted sites within the promoter region, give good insight in potential ZF-TF target selection criteria. Several (but not all) ZF-TFs targeted to the DNase I sensitive area were effective, and several (but not all) ZF-TF targeted to sites close to natural TF binding sites were active, including the most effective ZF-TF applied by the investigators. Of particular interest is that the one ZF-TF in this study that could both up- and downregulate gene expression (the 6-finger ZF-16 at position -369) was targeted within 20 bp of both a natural activator and a natural repressor binding site (Lund et al., 2004). The authors concluded that the exact site within the promoter region to which the ZF-TF is targeted is important to establish interaction with endogenous factors and result in the in vivo effect.

2. DNA-binding affinity Despite the fact that ZFP binding to naked DNA does not guarantee binding of the ZFP to the same sequences in vivo, the affinity by which the designed ZF-TF binds to DNA is an important parameter. A low Kd (indicating that the ZFP binds its target DNA with high affinity) is required for a ZF-TF to have an effect on its target gene (Beerli et al., 2000a; Graslund et al., 2005; Segal et al., 2004; Zhang et al., 2000). However, since the Kd is measured on naked DNA there is

4. Successes and Pitfalls of Engineered Transcription Factors

155

no strict relationship between measured Kd and endogenous gene-modulation. Zhang et al. (2000) designed reporter plasmids containing slight mutations in the zinc-finger recognition sites. They compared the affinity with which one of their engineered ZF-TFs bound to these sequences with the ability to induce the luciferase reporter gene. They observed a clear relation between affinity and gene induction—activation was most efficient with a Kd below 10 nM and relatively inefficient when the Kd exceeded 30 nM. Moreover, two ZF-TFs designed to bind to closely related sequences in promoter regions of the ErbB-1 and ErbB-2 genes were able to activate the target gene selectively, binding with a Kd around 1 nM, while the related, but unaffected promoter was bound with an affinity of about 10 nM (Beerli et al., 2000a). These and other studies, suggest that binding with a Kd value of about 1 nM is associated with occupancy of the target site sufficient for imposed gene control. Even so, Lund et al. (2004) showed that one of their ZF-TFs, which is bound in a relatively weak manner (50 nM), was more efficient in gene modulation than tighter binding ZF-TFd positioned at different sites in the promoter region (Fig. 4.5).

IV. CONCLUDING REMARKS AND FUTURE PERSPECTIVES Several classes of DNA-binding agents have been exploited for therapeutic gene modulation, given that DNA-binding agents can be exploited to both up- and downregulate gene expression (Uil et al., 2003). The targeting of DNA for downregulation of gene expression represents several advantages over targeting RNA. Generally, DNA-binding agents: (i) need to bind only two copies per cell and (ii) several genes can potentially be modulated simultaneously. For induction of gene expression, the direct targeting of DNA should lead to upregulation of all splice variants in the normal stoichiometric ratio, instead of affecting a single splice variant when cDNA is introduced. Engineered ZF-TFs are the most advanced DNA targeting agents and can be designed to target unique sequences in the human genome. ZF-repressors have been shown to act synergistically with siRNA in downregulation of gene expression (Kwon et al., 2005). The design of zinc-finger modules to bind specific DNA sequences has become fairly robust. However, whether the ZF-TF actually will bind to and act on chromatin in vivo is still difficult to predict. From the studies published so far, a few guidelines can be distilled to increase the chance of successful design. These include high-affinity binding of the ZF-TF to naked DNA and selecting target sequences that are located in an “accessible” region near the promoter or the transcriptional start site. Nucleosome position is the best-defined parameter influencing the accessibility of a ZF-TF to its target DNA. In absence of knowledge about positioned nucleosomes, DNase I or micrococcal digestion data and computational efforts to determine neighboring ALU repeat sequences

156

Visser et al.

or natural transcription-factor binding sites, can help in target selection. In the accompanying chapter (Verschure et al., Chapter 5, this issue), other parameters that may influence ZF-TF activity are discussed in detail, including the choice of effector domain. The modularity of ZF-TFs allows attachment of a wide variety of effector domains. These can cause temporary or permanent changes in gene expression (reviewed in this chapter), can be engineered to be constitutively active or inducible (Beerli et al., 2000b), or can even change gene function (Urnov et al., 2005). Using engineered ZF-TFs, expression modulation of a dozen endogenous genes has been achieved. Modulation of endogenous gene expression represents a novel, promising approach for the treatment of many different types of pathological conditions. In this respect, ZF-TFs are flexible tools for the specific regulation of endogenous genes. ZF-TFs have many possible applications. They can be used for fundamental research, development of bioassays for drug discovery (Liu et al., 2004), and in therapeutic applications. This makes these engineered proteins valuable and promising tools in the field of biomedical research.

Acknowledgments AEV is supported in part by The Netherlands Genomics Initiative (Horizon Breakthrough 050-71009) and Philip Morris Incorporated. PJV is supported by The Netherlands Organization for Scientific Research (NWO)(VIDI 2003/03921/ALW/016.041.311).

REFERENCES Agrawal, N., Dasaradhi, P. V., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., and Mukherjee, S. K. (2003). RNA interference: Biology, mechanism, and applications. Microbiol. Mol. Biol. Rev. 67, 657–685. Arnaud, P., and Feil, R. (2005). Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction. Birth Defects Res. C. Embryo. Today 75, 81–97. Ausio, J., Levin, D. B., De Amorim, G. V., Bakker, S., and Macleod, P. M. (2003). Syndromes of disordered chromatin remodeling. Clin. Genet. 64, 83–95. Bartsevich, V. V., and Juliano, R. L. (2000). Regulation of the MDR1 gene by transcriptional repressors selected using peptide combinatorial libraries. Mol. Pharmacol. 58, 1–10. Bartsevich, V. V., Miller, J. C., Case, C. C., and Pabo, C. O. (2003). Engineered zinc finger proteins for controlling stem cell fate. Stem Cells 21, 632–637. Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000a). Positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl. Acad. Sci. USA 97, 1495–1500. Beerli, R. R., Schopfer, U., Dreier, B., and Barbas, C. F., III (2000b). Chemically regulated zinc finger transcription factors. J. Biol. Chem. 275, 32617–32627. Beerli, R. R., Segal, D. J., Dreier, B., and Barbas, C. F., III. (1998). Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc

4. Successes and Pitfalls of Engineered Transcription Factors

157

finger proteins constructed from modular building blocks. Proc. Natl. Acad. Sci. USA 95, 14628–14633. Blaese, R. M., Culver, K. W., Miller, A. D., Carter, C. S., Fleisher, T., Clerici, M., Shearer, G., Chang, L., Chiang, Y., Tolstoshev, P., Greenblatt, J. J., Rosenberg, S. A., et al. (1995). T lymphocyte-directed gene therapy for ADA-SCID: Initial trial results after 4 years. Science 270, 475–480. Blancafort, P., Magnenat, L., and Barbas, C. F. (2003). Scanning the human genome with combinatorial transcription factor libraries. Nat. Biotechnol. 21, 269–274. Carmeliet, P., Ng, Y. S., Nuyens, D., Theilmeier, G., Brusselmans, K., Cornelissen, I., Ehler, E., Kakkar, V. V., Stalmans, I., Mattot, V., Perriard, J. C., Dewerchin, M., et al. (1999). Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat. Med. 5, 495–502. Cavazzana-Calvo, M., and Fischer, A. (2004). Efficacy of gene therapy for SCID is being confirmed. Lancet 364, 2155–2156. Chakravarthy, S., Park, Y. J., Chodaparambil, J., Edayathumangalam, R. S., and Luger, K. (2005). Structure and dynamic properties of nucleosome core particles. FEBS Lett. 579, 895–898. Cheng, K., Ye, Z., Guntaka, R. V., and Mahato, R. I. (2005). Biodistribution and hepatic uptake of triplex-forming oligonucleotides against type alpha1(I) collagen gene promoter in normal and fibrotic rats. Mol. Pharm. 2, 206–217. Collingwood, T. N., Urnov, F. D., and Wolffe, A. P. (1999). Nuclear receptors: Coactivators, corepressors and chromatin remodeling in the control of transcription. J. Mol. Endocrinol. 23, 255–275. Couzin, J. (2004). Molecular biology. RNAi shows cracks in its armor. Science 306, 1124–1125. Crossley, M., Ludwig, M., Stowell, K. M., De, V. P., Olek, K., and Brownlee, G. G. (1992). Recovery from hemophilia B Leyden: An androgen-responsive element in the factor IX promoter. Science 257, 377–379. Dai, Q., Huang, J., Klitzman, B., Dong, C., Goldschmidt-Clermont, P. J., March, K. L., Rokovich, J., Johnstone, B., Rebar, E. J., Spratt, S. K., Case, C. C., Kontos, C. D., et al. (2004). Engineered zinc finger-activating vascular endothelial growth factor transcription factor plasmid DNA induces therapeutic angiogenesis in rabbits with hindlimb ischemia. Circulation 110, 2467–2475. Datta, H. J., and Glazer, P. M. (2001). Intracellular generation of single-stranded DNA for chromosomal triplex formation and induced recombination. Nucleic Acids Res. 29, 5140–5147. de Kok, Y. J., van der Maarel, S. M., Bitner-Glindzicz, M., Huber, I., Monaco, A. P., Malcolm, S., Pembrey, M. E., Ropers, H. H., and Cremers, F. P. (1995). Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science 267, 685–688. Dickinson, L. A., Burnett, R., Melander, C., Edelson, B. S., Arora, P. S., Dervan, P. B., and Gottesfeld, J. M. (2004). Arresting cancer proliferation by small-molecule gene regulation. Chem. Biol. 11, 1583–1594. Dorsett, Y., and Tuschl, T. (2004). siRNAs: Applications in functional genomics and potential as therapeutics. Nat. Rev. Drug Discov. 3, 318–329. Drew, H. R. (1984). Structural specificities of five commonly used DNA nucleases. J. Mol. Biol. 176, 535–557. Dyson, H. J., and Wright, P. E. (2004). Unfolded proteins and protein folding studied by NMR. Chem. Rev. 104, 3607–3622. Edayathumangalam, R. S., Weyermann, P., Gottesfeld, J. M., Dervan, P. B., and Luger, K. (2004). Molecular recognition of the nucleosomal “supergroove.” Proc. Natl. Acad. Sci. USA 101, 6864–6869. Egger, G., Liang, G. N., Aparicio, A., and Jones, P. A. (2004). Epigenetics in human disease and prospects for epigenetic therapy. Nature 429, 457–463.

158

Visser et al.

Englander, E. W., and Howard, B. H. (1995). Nucleosome positioning by human Alu elements in chromatin. J. Biol. Chem. 270, 10091–10096. Everts, M., and Curiel, D. T. (2004). Transductional targeting of adenoviral cancer gene therapy. Curr. Gene Ther. 4, 337–346. Falke, D., Fisher, M., Ye, D., and Juliano, R. L. (2003). Design of artificial transcription factors to selectively regulate the pro-apoptotic bax gene. Nucleic Acids Res. 31, e10. Gabellini, D., Green, M. R., and Tupler, R. (2004). When enough is enough: Genetic diseases associated with transcriptional derepression. Curr. Opin. Genet. Dev. 14, 301–307. Gearhart, M. D., Dickinson, L., Ehley, J., Melander, C., Dervan, P. B., Wright, P. E., and Gottesfeld, J. M. (2005). Inhibition of DNA binding by human estrogen-related receptor 2 and estrogen receptor alpha with minor groove binding polyamides. Biochemistry 44, 4196–4203. Georgel, P. T. (2005). Chromatin potentiation of the hsp70 promoter is linked to GAGA-factor recruitment. Biochem. Cell Biol. 83, 555–565. Gilbert, J., Gore, S. D., Herman, J. G., and Carducci, M. A. (2004). The clinical application of targeting cancer through histone acetylation and hypomethylation. Clin. Cancer Res. 10, 4589–4596. Glover, D. J., Lipps, H. J., and Jans, D. A. (2005). Towards safe, non-viral therapeutic gene expression in humans. Nat. Rev. Genet. 6, 299–310. Gommans, W. G., Haisma, H. J., and Rots, M. G. (2005). Engineering zinc finger protein transcription factors: The therapeutic relevance of switching endogenous gene expression on or off at command. J. Mol. Biol., 354(3), 507–519. Gottesfeld, J. M., Melander, C., Suto, R. K., Raviol, H., Luger, K., and Dervan, P. B. (2001). Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides. J. Mol. Biol. 309, 615–629. Graslund, T., Li, X., Magnenat, L., Popkov, M., and Barbas, C. F., III. (2005). Exploring strategies for the design of artificial transcription factors: Targeting sites proximal to known regulatory regions for the induction of gamma-globin expression and the treatment of sickle cell disease. J. Biol. Chem. 280, 3707–3714. Hacein-Bey-Abina, S., Von, K. C., Schmidt, M., McCormack, M. P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C. S., Pawliuk, R., Morillon, E., Sorensen, R., Forster, A., et al. (2003). LMO2associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419. Jackson, A. L., Bartz, S. R., Schelter, J., Kobayashi, S. V., Burchard, J., Mao, M., Li, B., Cavet, G., and Linsley, P. S. (2003). Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637. Jouvenot, Y., Ginjala, V., Zhang, L., Liu, P. Q., Oshimura, M., Feinberg, A. P., Wolffe, A. P., Ohlsson, R., and Gregory, P. D. (2003). Targeted regulation of imprinted genes by synthetic zincfinger transcription factors. Gene Ther. 10, 513–522. Kawasaki, H., and Taira, K. (2004). Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature 431, 211–217. Kleinjan, D. A., and van Heyningen, V. (2005). Long-range control of gene expression: Emerging mechanisms and disruption in disease. Am. J. Hum. Genet. 76, 8–32. Korst, R. J., McElvaney, N. G., Chu, C. S., Rosenfeld, M. A., Mastrangeli, A., Hay, J., Brody, S. L., Eissa, N. T., Danel, C., and Jaffe, H. A. (1995). Gene therapy for the respiratory manifestations of cystic fibrosis. Am. J. Respir. Crit. Care Med. 151, S75–S87. Kubo, K., Sakamoto, A., Kobayashi, A., Rybka, Z., Kanno, Y., Nakagawa, H., and Takatsuji, H. (1998). Cys2/His2 zinc-finger protein family of petunia: Evolution and general mechanism of target-sequence recognition. Nucleic Acids Res. 26, 608–615. Kwon, H. S., Shin, H. C., and Kim, J. S. (2005). Suppression of vascular endothelial growth factor expression at the transcriptional and post-transcriptional levels. Nucleic Acids Res. 33, e74.

4. Successes and Pitfalls of Engineered Transcription Factors

159

Leung, R. K., and Whittaker, P. A. (2005). RNA interference: From gene silencing to gene-specific therapeutics. Pharmacol. Ther. 107, 222–239. Levitsky, V. G., Katokhin, A. V., Podkolodnaya, O. A., Furman, D. P., and Kolchanov, N. A. (2005). NPRD: Nucleosome positioning region database. Nucleic Acids Res. 33, D67–D70. Li, G., Levitus, M., Bustamante, C., and Widom, J. (2005). Rapid spontaneous accessibility of nucleosomal DNA. Nat. Struct. Mol. Biol. 12, 46–53. Li, G., and Widom, J. (2004). Nucleosomes facilitate their own invasion. Nat. Struct. Mol. Biol. 11, 763–769. Lin, E., and Nemunaitis, J. (2004). Oncolytic viral therapies. Cancer Gene Ther. 11, 643–664. Liu, P. Q., Morton, M. F., Reik, A., de la Rosa, R., Mendel, M. C., Li, X. Y., Case, C. C., Pabo, C. O., Moreno, V., Kempf, A., Pyati, J., and Shankley, N. P. (2004). Cell lines for drug discovery: Elevating target-protein levels using engineered transcription factors. J. Biomol. Screen. 9, 44–51. Liu, P. Q., Rebar, E. J., Zhang, L., Liu, Q., Jamieson, A. C., Liang, Y., Qi, H., Li, P. X., Chen, B., Mendel, M. C., Zhong, X., Lee, Y. L., et al. (2001). Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J. Biol. Chem. 276, 11323–11334. Liu, Q., Xia, Z., Zhong, X., and Case, C. C. (2002). Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J. Biol. Chem. 277, 3850–3856. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997). Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251–260. Lund, C. V., Blancafort, P., Popkov, M., and Barbas, C. F., III. (2004). Promoter-targeted phage display selections with preassembled synthetic zinc finger libraries for endogenous gene regulation. J. Mol. Biol. 340, 599–613. Magnenat, L., Blancafort, P., and Barbas, C. F., III. (2004). In vivo selection of combinatorial libraries and designed affinity maturation of polydactyl zinc finger transcription factors for ICAM-1 provides new insights into gene regulation. J. Mol. Biol. 341, 635–649. Melander, C., Burnett, R., and Gottesfeld, J. M. (2004). Regulation of gene expression with pyrroleimidazole polyamides. J. Biotechnol. 112, 195–220. Moore, M., Choo, Y., and Klug, A. (2001). Design of polyzinc finger peptides with structured linkers. Proc. Natl. Acad. Sci. USA 98, 1432–1436. Morris, K. V., Chan, S. W., Jacobsen, S. E., and Looney, D. J. (2004). Small interfering RNAinduced transcriptional gene silencing in human cells. Science 305, 1289–1292. O’Neill, M. J. (2005). The influence of non-coding RNAs on allele-specific gene expression in mammals. Hum. Mol. Genet. 14(1), R113–R120. Pavletich, N. P., and Pabo, C. O. (1991). Zinc finger-DNA recognition: Crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809–817. Pavletich, N. P., and Pabo, C. O. (1993). Crystal structure of a five-finger GLI-DNA complex: New perspectives on zinc fingers. Science 261, 1701–1707. Peisach, E., and Pabo, C. O. (2003). Constraints for zinc finger linker design as inferred from X-ray crystal structure of tandem Zif268-DNA complexes. J. Mol. Biol. 330, 1–7. Peng, Z. (2005). Current status of gendicine in China: Recombinant human Ad-p53 agent for treatment of cancers. Hum. Gene Ther. 16, 1016–1027. Qiu, S., Adema, C. M., and Lane, T. (2005). A computational study of off-target effects of RNA interference. Nucleic Acids Res. 33, 1834–1847. Rajagopalan, S., Mohler, E. R., III, Lederman, R. J., Mendelsohn, F. O., Saucedo, J. F., Goldman, C. K., Blebea, J., Macko, J., Kessler, P. D., Rasmussen, H. S., and Annex, B. H. (2003). Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: A phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 108, 1933–1938.

160

Visser et al.

Rebar, E. J. (2004). Development of pro-angiogenic engineered transcription factors for the treatment of cardiovascular disease. Expert. Opin. Invest. Drugs 13, 829–839. Rebar, E. J., Huang, Y., Hickey, R., Nath, A. K., Meoli, D., Nath, S., Chen, B., Xu, L., Liang, Y., Jamieson, A. C., Zhang, L., Spratt, S. K., et al. (2002). Induction of angiogenesis in a mouse model using engineered transcription factors. Nat. Med. 8, 1427–1432. Ren, D., Collingwood, T. N., Rebar, E. J., Wolffe, A. P., and Camp, H. S. (2002). PPARgamma knockdown by engineered transcription factors: Exogenous PPARgamma2 but not PPARgamma1 reactivates adipogenesis. Genes Dev. 16, 27–32. Sadeghi, H., and Hitt, M. M. (2005). Transcriptionally targeted adenovirus vectors. Curr. Gene Ther. 5, 411–427. Saunthararajah, Y., Hillery, C. A., Lavelle, D., Molokie, R., Dorn, L., Bressler, L., Gavazova, S., Chen, Y. H., Hoffman, R., and DeSimone, J. (2003). Effects of 5-aza-20 -deoxycytidine on fetal hemoglobin levels, red cell adhesion, and hematopoietic differentiation in patients with sickle cell disease. Blood 102, 3865–3870. Scacheri, P. C., Rozenblatt-Rosen, O., Caplen, N. J., Wolfsberg, T. G., Umayam, L., Lee, J. C., Hughes, C. M., Shanmugam, K. S., Bhattacharjee, A., Meyerson, M., and Collins, F. S. (2004). Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1892–1897. Segal, D. J. (2002). The use of zinc finger peptides to study the role of specific factor binding sites in the chromatin environment. Methods 26, 76–83. Segal, D. J., Beerli, R. R., Blancafort, P., Dreier, B., Effertz, K., Huber, A., Koksch, B., Lund, C. V., Magnenat, L., Valente, D., and Barbas, C. F., III (2003). Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry 42, 2137–2148. Segal, D. J., Goncalves, J., Eberhardy, S., Swan, C. H., Torbett, B. E., Li, X., and Barbas, C. F., III. (2004). Attenuation of HIV-1 replication in primary human cells with a designed zinc finger transcription factor. J. Biol. Chem. 279, 14509–14519. Sera, T., and Uranga, C. (2002). Rational design of artificial zinc-finger proteins using a nondegenerate recognition code table. Biochemistry 41, 7074–7081. Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., and Williams, B. R. (2003). Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5, 834–839. Snove, O., Jr., and Holen, T. (2004). Many commonly used siRNAs risk off-target activity. Biochem. Biophys. Res. Commun. 319, 256–263. Snowden, A. W., Gregory, P. D., Case, C. C., and Pabo, C. O. (2002). Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo.. Curr. Biol. 12, 2159–2166. Snowden, A. W., Zhang, L., Urnov, F., Dent, C., Jouvenot, Y., Zhong, X., Rebar, E. J., Jamieson, A. C., Zhang, H. S., Tan, S., Case, C. C., Pabo, C. O., et al. (2003). Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors. Cancer Res. 63, 8968–8976. Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., John, M., Kesavan, V., et al. (2004). Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173–178. Tachikawa, K., Schroder, O., Frey, G., Briggs, S. P., and Sera, T. (2004). Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins. Proc. Natl. Acad. Sci. USA 101, 15225–15230. Takatsuji, H. (1998). Zinc-finger transcription factors in plants. Cell Mol. Life Sci. 54, 582–596. Takatsuji, H., and Matsumoto, T. (1996). Target-sequence recognition by separate-type Cys2/His2 zinc finger proteins in plants. J. Biol. Chem. 20(271), 23368–23373. Tan, S., Guschin, D., Davalos, A., Lee, Y. L., Snowden, A. W., Jouvenot, Y., Zhang, H. S., Howes, K., McNamara, A. R., Lai, A., Ullman, C., Reynolds, L., et al. (2003). Zinc-finger

4. Successes and Pitfalls of Engineered Transcription Factors

161

protein-targeted gene regulation: Genomewide single-gene specificity. Proc. Natl. Acad. Sci. USA 100, 11997–12002. Thorvaldsen, J. L., Duran, K. L., and Bartolomei, M. S. (1998). Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes Dev. 12, 3693–3702. Uil, T. G., Haisma, H. J., and Rots, M. G. (2003). Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities. Nucleic Acids Res. 31, 6064–6078. Urban-Klein, B., Werth, S., Abuharbeid, S., Czubayko, F., and Aigner, A. (2005). RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther. 12, 461–466. Urnov, F. D. (2002). A feel for the template: Zinc finger protein transcription factors and chromatin. Biochem. Cell Biol. 80, 321–333. Urnov, F. D. (2003). Chromatin remodeling as a guide to transcriptional regulatory networks in mammals. J. Cell Biochem. 88, 684–694. Urnov, F. D., Miller, J. C., Lee, Y. L., Beausejour, C. M., Rock, J. M., Augustus, S., Jamieson, A. C., Porteus, M. H., Gregory, P. D., and Holmes, M. C. (2005). Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651. Van Eenennaam, A. L., Li, G., Venkatramesh, M., Levering, C., Gong, X., Jamieson, A. C., Rebar, E. J., Shewmaker, C. K., and Case, C. C. (2004). Elevation of seed alpha-tocopherol levels using plant-based transcription factors targeted to an endogenous locus. Metab. Eng. 6, 101–108. Verma, I. M., and Weitzman, M. D. (2005). Gene therapy: Twenty-first century medicine. Annu. Rev. Biochem. 74, 711–738. Verschure, P. J., Visser, A. E., and Rots, M. G. (2005). Step out of the groove: Epigenetic gene control systems and engineered transcription factors. Adv. Gen. 56. Villard, J. (2004). Transcription regulation and human diseases. Swiss Med. Wkly. 134, 571–579. Wannenes, F., Ciafre, S. A., Niola, F., Frajese, G., and Farace, M. G. (2005). Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo. Cancer Gene Ther. 12, 926–934. Xia, H., Mao, Q., Eliason, S. L., Harper, S. Q., Martins, I. H., Orr, H. T., Paulson, H. L., Yang, L., Kotin, R. M., and Davidson, B. L. (2004). RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med. 10, 816–820. Xu, D., Ye, D., Fisher, M., and Juliano, R. L. (2002). Selective inhibition of P-glycoprotein expression in multidrug-resistant tumor cells by a designed transcriptional regulator. J. Pharmacol. Exp. Ther. 302, 963–971. Yla-Herttuala, S., Markkanen, J. E., and Rissanen, T. T. (2004). Gene therapy for ischemic cardiovascular diseases: Some lessons learned from the first clinical trials. Trends Cardiovasc. Med. 14, 295–300. Yuan, G. C., Liu, Y. J., Dion, M. F., Slack, M. D., Wu, L. F., Altschuler, S. J., and Rando, O. J. (2005). Genome-scale identification of nucleosome positions in S. cerevisiae. Science 309, 626–630. Zhang, L., Spratt, S. K., Liu, Q., Johnstone, B., Qi, H., Raschke, E. E., Jamieson, A. C., Rebar, E. J., Wolffe, A. P., and Case, C. C. (2000). Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J. Biol. Chem. 275, 33850–33860.

This page intentionally left blank

5 Step out of the Groove:

Epigenetic Gene Control Systems and Engineered Transcription Factors Pernette J. Verschure,* Astrid E. Visser,*,† and Marianne G. Rots‡ *Swammerdam Institute for Life Sciences, BioCentrum Amsterdam University of Amsterdam, 1098SM Amsterdam The Netherlands † New Address: Department of Molecular Genetics, Leiden Institute of Chemistry University of Leiden, 2300 RA Leiden, The Netherlands ‡ Therapeutic Gene Modulation Groningen University Institute for Drug Exploration University of Groningen, The Netherlands

I. Influence of Epigenetic Mechanisms on Gene Expression A. Nucleosomal level B. Large-scale chromatin level C. Nuclear level D. Human diseases and epigenetic gene control mechanisms II. How Do ZF-TFS Modulate Epigenetic Gene Regulation? A. Activation effector domains B. Comparison of the effect between different activation domains C. Repression effector domains D. Comparison of the effect between different repression domains III. Epigenetic Aspects to Consider for ZF-TF Approaches A. Functional chromatin status versus ZF-TF control B. Target definition C. Specificity D. Efficiency E. Concluding remarks Acknowledgments References *

Corresponding author: [email protected]

Advances in Genetics, Vol. 56 Copyright 2006, Elsevier Inc. All rights reserved.

0065-2660/06 $35.00 DOI: 10.1016/S0065-2660(06)56005-5

164

Verschure et al.

ABSTRACT At the linear DNA level, gene activity is believed to be driven by binding of transcription factors, which subsequently recruit the RNA polymerase to the gene promoter region. However, it has become clear that transcriptional activation involves large complexes of many different proteins, which not only directly recruit components of the transcription machinery but also affect the DNA folding. Such proteins, including various chromatin-modifying enzymes, alter among other processes nucleosome positioning and histone modifications and are potentially involved in changing the overall structure of the chromatin and/or the position of chromatin in the nucleus. These epigenetic regulatory features are now known to control and regulate gene expression, although the molecular mechanisms still need to be clarified in more detail. Several diseases are characterized by aberrant gene-expression patterns. Many of these diseases are linked to dysregulation of epigenetic gene-regulatory systems. To interfere with aberrant gene expression, a novel approach is emerging as a disease therapy, involving engineered transcription factors. Engineered transcription factors are based on, for example, zinc-finger proteins (ZFP) that bind DNA in a sequence-specific manner. Engineered transcription factors based on ZFP are fused to effector domains that function to normalize disrupted geneexpression levels. Zinc-finger proteins most likely also influence epigenetic regulatory systems, such as the complex set of chemical histone and DNA modifications, which control chromatin compaction and nuclear organization. In this chapter, we review how epigenetic regulation systems acting at various levels of packaging the genome in the cell nucleus add to gene-expression control at the DNA level. Since an increasing number of diseases are described to have a clear link to epigenetic dysregulation, we here highlight 10 examples of such diseases. In the second part, we describe the different effector domains that have been fused to ZFPs and are capable of activating or silencing endogenous genes, and we illustrate how these effector domains influence epigenetic control mechanisms. Finally, we speculate how accumulating knowledge about epigenetics can be exploited to make such zinc-finger–transcription factors (ZF-TF) even more effective. ß 2006, Elsevier Inc.

I. INFLUENCE OF EPIGENETIC MECHANISMS ON GENE EXPRESSION Higher-order genomes contain two types of information—the genetic information, which is based on the well-known double helix consisting of DNA sequences and epigenetic information, which forms a regulatory layer on top of the DNA sequence information. The epigenetic information determines which genes are silenced and which genes can be activated at the right time in the right cells.

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

165

Epigenetic information is coded by local DNA methylation as well as by the packaging of DNA into nucleosomes and chromatin. These packaging phenomena act at different levels, as illustrated in Fig. 5.1: (1) DNA and nucleosomal, (2) large-scale chromatin, and (3) nuclear level. These three levels of epigenetic genome packaging can be considered as three interacting systems. Epigenetic gene-control mechanisms are required for genome (re)programing during development to obtain tissue-specific gene-expression patterns (Jaenisch and Bird, 2003). Moreover, epigenetic control systems involve the maintenance of defined heritable gene expression patterns (Cavalli and Paro, 1999). Strictly, the term “epigenetic” refers to meiotically or mitotically inheritable features. However, particular histone modifications are also involved in short-term gene regulation. This has led to a generalization of the term epigenetic (epi ¼ besides) referring to any (genomic) regulatory feature that does not strictly depend on regulation at the DNA sequence level. In general, epigenetic mechanisms control the regulation of several nuclear processes, such as transcription, replication, DNA repair, recombination, and genome stability. In contrast to the DNA sequence code that remains identical in each cell and

Figure 5.1. Epigenetic genome packaging aspects. The genetic code (DNA) of eukaryotic cells is packaged in chromatin and stored in the cell nucleus. DNA is folded around a protein complex of histones to form nucleosomes. Arrays of nucleosomes form 30-nm fibers and subsequently higher-order chromatin structures. In addition, this figure illustrates where epigenetic regulatory information can be added: (A) DNA is locally modified by, for instance, DNA methylation; histones are chemically modified and nucleosomes can be positioned; (B) chromatin is organized at the large scale, such as different chromatin compaction states; and (C) at the nuclear level, genes and gene loci occupy preferred positions within the nucleus. These levels of epigenetic genome packaging can be considered as interacting systems. This picture is adapted from an illustration kindly provided by Professor J. Hansen, Department of Biochemistry and Molecular Biology, Colorado State University, USA. From “The Annual Review of Biophysics and Biomolecular Structure,” Vol. 31, pp. 361–392 by Annual Reviews (2002) www. annualreviews.org.

166

Verschure et al.

can therefore be considered as “read-only,” the epigenetic code can be read, rewritten, and also erased. Because of this reversible character, modulation of gene expression via epigenetic control mechanisms are increasingly viewed in connection with promising tools to treat or possibly cure gene dysregulation as occurs in conjunction with several diseases (Egger et al., 2004). A promising approach to interfere with epigenetic control mechanisms to modulate gene expression is by engineered transcription factors. These are engineered DNA binding modules (such as zinc-finger proteins [ZFP]) that bind DNA in a sequence-specific manner, fused to an effector domain that can up- or downregulate gene expression. We refer to these proteins as zinc-finger–based transcription factors (ZF-TFs). For a dozen of genes, a variety of studies have shown the potency of this approach. These studies are reviewed in detail in the accompanying Chapter 4 by Visser et al. In the present chapter, we give an overview how the genome is packaged in the cell nucleus and describe how each level of genomic organization affects epigenetic gene control. Some examples are provided with respect to the manner by which these levels of epigenetic genome organization are related to several diseases. Next we depict how ZF-TFs act in relation to epigenetics and indicate how we can start to apply knowledge about epigenetic gene control mechanisms for successful approaches to disease therapy.

A. Nucleosomal level The basic level of eukaryotic chromatin structure (Fig. 5.1) is the nucleosome, which consists of approximately 150 base pairs DNA wrapped around a core histone octamer—2 copies of each of the histones H2A, H2B, H3, and H4 (Luger et al., 1997). The nucleosome is sealed with a single molecule of linker histone H1. The crystal structure of a nucleosome core particle has provided considerable insight into the protein–protein and protein–DNA interactions that govern nucleosome structure (Luger et al., 1997).

1. Nucleosomal packaging Wrapping of DNA around the nucleosome changes the accessibility of DNA to regulatory proteins (including ZF-TFs) (Chapter 4 and references; Urnov, 2002, 2003). In yeast, many nucleosomes occupy precise positions with respect to regulatory sequences (Yuan et al., 2005). Such positioning is believed to provide a subtle mechanism for gene regulation. This is illustrated by the experimental shifting of a positioned nucleosome with respect to the TATA box of the PHO5 gene by as little as two to three base pairs. This shift leads to rotation of the TATA box on the nucleosome surface, rendering it poorly accessible to, for instance, the transcription factor TFIID. The affected gene can still be

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

167

activated, but this now depends on acetylation of histone H4 and the protein Bdf1, which interact with both acetylated histones and TFIID (Martinez-Campa et al., 2004). In mammalian cells, however, fewer nucleosomes are believed to be precisely positioned. Even so, a number of mostly in vitro observations formed the basis of the “classic determinants” of nucleosomal chromatin structure (Gilbert and Allan, 2001; Sun et al., 2001; Wallrath and Elgin, 1995; Weintraub and Groudine, 1976). Inactive chromatin consists of nucleosomes that: (i) are arranged in a more regular nucleosome array, (ii) have a shorter length of the linker region between the nucleosomes, and (iii) contain DNA that is highly protected against digestion with nucleases such as DNase I, micrococcal nuclease, and restriction enzymes. In contrast, chromatin in which high levels of gene expression are occurring consists of less regular nucleosomal packaging, allowing nonhistone proteins to bind to chromatin and to recruit chromatin remodeling and modification factors. It is thus generally considered that factors at the nucleosomal level influence packaging of chromatin, accessibility of the DNA, hence transcription activity. The ability of regulatory factors to bind at the nucleosomal level will substantially influence the ability of engineered ZF-TF to reach their target.

2. Epigenetic gene control via modifications at the nucleosomal level Epigenetic gene control mechanisms are thought to influence nucleosomal packaging, thereby controlling the probability of molecular interactions with DNA (Cosgrove et al., 2004; Felsenfeld and Groudine, 2003; Jenuwein and Allis, 2001; Lachner and Jenuwein, 2002; Turner, 2002). Examples of such epigenetic gene control systems are DNA methylation, posttranslational histone modifications, histone variant incorporation, and RNA-associated gene silencing. Methylation of cytosine in CpG dinucleotides is a widespread modification in the human genome, associated with transcriptional silencing and the formation of heterochromatin (Salozhin et al., 2005). DNA methylation can affect gene expression directly and indirectly. For instance, some transcription factors, such as Sp1, can interact only with nonmethylated DNA sequences. Methylation of cytosine abolishes interaction of transcription factors with DNA, resulting in gene repression (Clark et al., 1997). On the other hand, proteins containing a methyl-DNA–binding domain (MBD), such as methylCpG–binding protein 2 (MeCP2), can specifically recognize methylated CpG dinucleotides and are involved in changing chromatin structure. Specific residues on histones are posttranslationally modified by phosphorylation, acetylation, and methylation as well as by other less well-known

168

Verschure et al.

histone modifications such as ubiquitination, sumoylation, ADP ribosylation, glycosylation, biotinylation, and carbonylation (Margueron et al., 2005). Most described residues that are modified are located at the flexible N-terminal histone tail that protrudes out of the nucleosome (Cosgrove et al., 2004). Modifications located at the globular core of histones have also been described (Cosgrove et al., 2004). The histone modifications are termed according to the system that first identifies the histone, then the amino acid modified, and the group that is added. For example, acetylation of histone H4 at lysine 8 is termed H4K8ac. The combination of modified residues, rather than the modification of a single residue, determines the conformation of chromatin and thereby the regulation of gene expression. For instance, the combination of H4K8ac, H3K14ac, and H3S10p are often associated with transcription (Zhang and Reinberg, 2001). Conversely, H3K9me3, in combination with the lack of acetylation of H3 and H4, correlates with transcriptional repression (Fischle et al., 2003a; Zhang and Reinberg, 2001). A histone modification of one residue can also affect the modification of another, even when they are located on different histones (e.g., histone deacetylation is a prerequisite for histone H3 methylation at lysine 9) (Nakayama et al., 2001; Rea et al., 2000; Rice and Allis, 2001; Turner, 2002). Histone modifications are driven by defined enzymes (e.g., histone acetylation is upregulated by histone acetyl transferases [HATs] and downregulated by histone deacetylases [HDACs]). Furthermore, multiple histone-modifying enzymes (e.g., the methyltransferases [HMT] that drive histone methylation) are able to modify the same lysine, resulting in different lysine states (mono-, di-, or trimethylated). In fact, the degree of lysine methylation determines the functional chromatin status, as level, as is explained in a later section (Rice et al., 2003). Modification of histone residues provides binding sites for chromatinassociated proteins. Proteins containing a bromodomain can specifically interact with acetylated histone residues, while proteins that contain a chromodomain can specifically bind methylated histone residues (Daniel et al., 2005; Fischle et al., 2003b). Different histone-modifying enzymes are targeted to different chromosomal domains, and the effect of a histone modification at a given locus depends on the presence of interacting partners at that locus. For instance, specific HMT direct mono- and dimethylated histone H3 at lysine 9 to transiently inactive chromatin, whereas the bulk of trimethylated histone H3 at lysine 9 is directed to permanently silenced chromatin (Perez-Burgos et al., 2004; Peters et al., 2003). It has also been suggested that histone modifications can alter the charge of the histones and thereby the interaction of the histones with DNA, which could result in condensation or decondensation of chromatin (Margueron et al., 2005). In summary, histone modifications are considered to directly link transcriptional regulation with changes in chromatin structure (Wang et al., 2004b).

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

169

Another important mechanism by which chromatin can be remodeled is the replacement of major histones with specific histone variants. Several core histone variants have been reported that are associated with a particular function. For instance, the incorporation of histone variants H3.3 and H2A.Z is thought to facilitate gene activity due to a less packaged chromatin structure, possibly mediated by histone modifications (Henikoff et al., 2004). Also histone variants have been included in the removal of epigenetic marks on modified canonical histones (for further reading, see Henikoff et al., 2004). The concept is that an epigenetic mark, such as a modified histone, acts as a docking site for an effector protein that initiates a distinct downstream event (Wang et al., 2004a,b). Emerging evidence suggests that different forms of epigenetic marks, DNA methylation, histone modifications, histone variants, and small nuclear RNAs or RNA interference, act in a concerted action (Allshire, 2002; Fuks et al., 2000; Mutskov et al., 2002; Volpe et al., 2002). A link between histone modifications and DNA methylation is formed by methyl-CpG–binding proteins such as MeCP2 (Fuks et al., 2000). MeCP2 recruits histone deacetylation complexes to deacetylate histone tails to allow subsequent histone methylation. The discovery that small RNAs associated with the RNA interference pathway are involved in chromatin compaction, thereby acting at the DNA or chromatin level, has revealed a completely new aspect of silent chromatin (Allshire, 2002; Martienssen, 2003; Reinhart and Bartel, 2002; Verdel et al., 2004). Knowledge about how small RNAs target silencing proteins and histone-modifying enzymes to heterochromatin is not yet available (for further reading, see Kim, 2005). The mechanisms by which ZF-TFs (and their respective activation and repression domains) possibly interfere with such epigenetic gene control systems are discussed in detail in Section II of this chapter.

B. Large-scale chromatin level Packaging of the DNA around the nucleosome and the molecular mechanisms acting at this level are well understood, compared with subsequent “higherorder” levels of organization. The nucleosomal array (also called the 10-nm fiber consisting of 2 11-nm sized nucleosomes) folds into 30-nm fibers (Dorigo et al., 2004) (Fig. 5.1). It is evident from electron and light microscopy that higher-order arrangements of 30-nm fibers exist, such as 100–130-nm so-called “chromonema fibers” (Belmont and Bruce, 1994). Such fibers do not randomly spread throughout the cell nucleus but form a discrete chromosome territory (Cremer and Cremer, 2001; Visser and Aten, 1999). Chromosome territories are structures consisting of condensed chromatin and a considerable amount of interchromatin space (Verschure et al., 1999; Visser et al., 2000). The mechanisms that establish and maintain functional chromatin domains in relation to

170

Verschure et al.

gene expression are under active investigation but remain ill-understood. Here we describe some general features of chromatin domains and of large-scale chromatin organization that are thought to determine functional aspects of gene regulation.

1. Chromatin domains bordered by boundaries Chromatin consists of different functional domains, the most well known are euchromatin, constitutive heterochromatin, and facultative heterochromatin, although a much wider range of chromatin types exists (Craig, 2005; Dillon, 2004). Euchromatic regions are a mixture of actively transcribed chromatin and potentially active chromatin interspersed with transiently silenced gene loci. Facultative heterochromatin describes a permissive chromatin environment that is subject to gene silencing, for instance, after differentiation. Constitutive heterochromatin represents condensed chromatin, generally associated with telomeres and pericentric regions of chromosomes. Constitutive heterochromatin consists predominantly of repetitive sequences, related to transposable elements and retroviruses, and is typically gene poor although not devoid of genes. Transitions between chromatin states are tightly linked to changes in gene activity and are causally related to changes in covalent modifications of core histone proteins. Division of the genome into distinct functional chromatin domains is suggested to represent a key aspect of gene control. The functional state of chromatin domains characterized, for instance, by the histone-modification state, can spread over tens of kilobase pairs (in yeast) (Hall et al., 2002; Noma et al., 2001). This is exemplified by the silent mating-type loci in yeast where histone H3 methylation at lysine 9 is enriched over a stretch of 20 kilobase pairs (Hall et al., 2002) (Fig. 5.2). In contrast, transcriptionally active regions flanking the silent locus are depleted of histone H3 methylation at lysine 9. Specialized DNA boundary elements have been identified to define the borders between adjacent chromatin domains acting as barriers against effects of enhancer and silencer elements from neighboring chromatin regions (Byrd and Corces, 2003; Labrador and Corces, 2002). Mutation of such boundary elements was shown to result in propagation of histone H3 methylation at lysine 9 into surrounding euchromatin regions, further supporting the idea that spreading of the functional state in cis is bordered by such boundary elements (Hall et al., 2002).

2. Conformational changes in large-scale chromatin structure Changes in chromatin structure are tightly related to changes in gene activity (Horn and Peterson, 2002). Belmont and colleagues were the first to show in living cells that a transcriptional activator can change large-scale chromatin

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

171

Figure 5.2. Division of the genome into functional chromatin domains. Division of the genome into distinct functional chromatin domains is suggested to represent a key aspect of gene control. The functional state of chromatin domains, such as the histonemodification state, can spread over tens of kilobases within the domain. This is exemplified by the silent mating type loci in yeast. A defined epigenetic status (representing chromatin that is transcriptionally inactive) is found over a stretch of 20 kb (marked by arrowheadline) that is bordered by “boundaries” (marked by arrows) consisting of inverted repeats. Enrichment of histone H3 methylation at lysine 9 (light gray) is observed within the transcriptionally inactive chromatin domain, whereas the transcriptionally active regions flanking the domain are depleted of histone H3 methylation at lysine 9 but enhanced of histone H3 methylated at lysine 4 (dark gray). This figure is adapted from Hall et al. (2002). Science 297, 215–218.

folding and modify nuclear positioning of chromosome loci. They created cell lines that express a megabase pair-sized, amplified chromosome region containing a tandem array of 256 lac operator sequences. This region is visualized by a lac repressor eGFP-tagged fusion protein (Robinett et al., 1996). A dramatic extension of the GFP-tagged chromatin domain into fibers with a diameter of 25–40 mm could be observed when the lac operator region was manipulated with a transcriptional activator (i.e., VP16 fused to the eGFP lac repressor) (Robinett et al., 1996; Tumbar and Belmont, 2001; Tumbar et al., 1999). Direct in vivo visualization of chromatin fibers upon activation showed that the nonactivated locus is preferentially positioned close to the nuclear periphery (a heterochromatin-rich region with little gene activity, as described in Section I.C). Upon VP16 targeting, the locus moves into the nuclear interior (a region with more euchromatin containing gene activity) (Tumbar and Belmont, 2001). These studies, however, showed that the observed chromatin decondensation is not sufficient for transcriptional activation. Transcriptional activity of the domain

172

Verschure et al.

was not enhanced, and when using drugs to inhibit transcription the decondensed configuration was not affected (Tumbar et al., 1999). Studies performed by the McNally group show light microscopical observations of changes in the large-scale chromatin structure of a natural chromosome region consisting of a tandem array of about 200 genes controlled by the glucocorticoid receptor (GR) (Muller et al., 2001). In this system, in response to GR the chromatin becomes decondensed, and gene activity is induced. These observations suggest that chromatin decondensation is not necessarily linked with gene activity but at least the chromatin is prepared to become active. We demonstrated chromatin condensation (instead of decondensation as described in the previous paragraph) of the large-scale chromatin structure of a chromosomal array, by targeting heterochromatin protein 1 (HP1). HP1 is a protein known to be involved in epigenetic gene silencing (Brink et al., 2006; Verschure et al., 2005) (Fig. 5.3). Upon targeting HP1, as a lac repressor eGFP tagged fusion protein to a lac-operator containing, amplified chromosome region in an unusually extended “euchromatin-like” configuration, we observed distinct heterochromatinization of the region. The chromatin structure after HP1 targeting appeared as a collection of a small number of distinct intensely labeled subdomains of approximately 0.4-mm diameter. This suggests a local condensation of the large-scale chromatin structure. Moreover, HP1 targeting resulted in recruitment of additional silencers, such as histone methyltransferase SETDB1, trimethylation of histone H3 at lysine 9, and endogenous HP1. These data demonstrate that HP1 binding at a defined chromatin region is sufficient to induce heterochromatinization of that region. These systems illustrate in living cells that targeting of regulatory proteins involved in gene activation causes large-scale chromatin decondensation required to poise the chromatin for transcription, whereas targeting of silencing proteins causes large-scale chromatin compaction. Of interest, GR used in the studies of the McNally group, as targeting protein to a natural chromatin array controlled by GR, is a natural zinc-finger protein. Thus one might expect that engineered ZF-TF will also influence both the chromatin configuration and the gene activity of the targeted gene. It will be interesting to use ZF-TF as tools to further scrutinize the molecular mechanisms behind the relationship between chromatin configuration and gene activity.

3. Spatial relationship: Within or outside compact chromatin domains Gene activity, as well as other nuclear processes, such as DNA replication, are related to the location of the gene locus with respect to compact chromatin structures—a gene “burried” inside a compact chromatin domain is generally inactive, whereas extrusion of a gene outside of compact chromatin, such as by

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

173

Figure 5.3. Targeting of an epigenetic gene regulatory protein involved in gene silencing induces local compaction of the chromatin structure. Chromatin structure is manipulated and visualized in living cells via the targeting of lac repressor GFP tagged fusion proteins to an integrated lac operator repeat. In the control cells, lac repressor tagged GFP visualizes the chromatin domain as a diffuse large region. Upon targeting HP1 to the chromosomal array as lac repressor GFP tagged HP1 fusion protein, the chromatin is compacted (more intense, less diffuse signal) and trimethylation of histone H3 at lysine 9 is enhanced at the array (accumulation of red signal at green domain). Threedimensional images were recorded; images represent individual mid-nuclear optical sections. The chromosomal array in a control nucleus (A1) and HP1 targeted nucleus (B1) is shown. The arrow in A1 and B1 points to the chromosomal array. A2 and B2 represent the chromatin structure and A3 and B3 trimethylation of histone H3 at lysine 9. Bar in A1 represents 2 mm.

looping out, is associated with gene activity. Actively regulated sites, such as transcription sites (Cmarko et al., 1999; Verschure et al., 1999, 2002) and sites of DNA replication (Jaunin et al., 2000) occur concentrated at the surface of compact chromatin domains. Noteworthy also gene loci where Polycomb-group proteins bind (Polycomb-group proteins are a heterogeneous class of polypeptides involved in heritable gene silencing) are found to occur at such surfaces of compact chromatin domains (Cmarko et al., 2003). Taken together, “active” chromatin structures most likely present fine chromatin loops extending away from more condensed chromatin domains (Cmarko et al., 1999, 2003;

174

Verschure et al.

Verschure et al., 1999). This looping-out may be dynamically controlled. During DNA replication, the “active” sites internalize into the interior of the condensed chromatin within minutes (Jaunin et al., 2000). Also, work of Mahy et al. (2002a,b) showed that a ubiquitously expressed gene often colocalized with fine chromatin structures of the chromosome territory, whereas the adjacent noncoding DNA is positioned frequently within compact chromatin (Fig. 5.4A). The location of actively regulated sites outside of compact chromatin probably represents a general feature of gene regulation. We addressed the issue whether compact chromatin provides an environment inaccessible to chromatin proteins or factors of the transcription machinery (Verschure et al., 2003). We asked whether compact chromatin domains in nuclei of living cells are penetrated by inert dextrans of various molecular sizes. Our data demonstrated that compact chromatin domains visualized by histone H2B GFP are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. This finding indicates that steric hindrance of compact chromatin is not responsible for positioning of actively transcribed loci outside condensed chromatin or conversely involved in keeping nonexpressed loci inside condensed chromatin. There are now many observations showing that giant chromatin loops with transcriptionally active genes can extent beyond their chromosome territory, at a scale of several microns (Chambeyron and Bickmore, 2004; Volpi et al., 2000; Williams et al., 2002). For example, in human fibroblasts the MHC class II gene cluster consisting of several megabase pairs of DNA is more frequently relocated away from the main body of chromosome 6 upon gene induction (Volpi et al., 2000). Looping out of chromatin may mark out regions that have the potential for transcription, given a correct transcription-factor environment. Versteeg and coworkers presented evidence that the human genome contains at least 30 chromosomal domains characterized by high-gene density and high-gene-expression levels in a variety of cell types (Caron et al., 2001; Versteeg et al., 2003). Such domains of many megabase pairs, called regions of increased gene expression (RIDGES), might coincide with large chromatin loops that loop out of their chromosome territories or out of compact chromatin domains. If looping out of a compact chromatin domain correlates with the places where active gene expression is going on, one might speculate that the effect of engineered ZF-TFs will also depend on the positioning of the target gene with respect to the chromatin compaction status. Therefore, we might also speculate that using ZF-TFs to upregulate a silent gene, which is in a compact chromatin, heterochromatin-like environment is not easy. On the other hand, compact heterochromatin domains are shown to be accessible for large transcription complexes and such domains are thus also accessible for ZF-TFs. However, accessibility for compact chromatin does not imply that ZF-TF–DNA binding can be achieved.

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

175

C. Nuclear level There is considerable evidence that not only the folding of the chromatin, but also the architecture of the nucleus is closely related to genome function (Spector, 2001; Verschure, 2004) (Fig. 5.1). The position of a gene inside the nucleus is shown to be related to the functionality of the gene—some areas in the nucleus are repressive for gene activity, whereas others promote or even boost transcription (Verschure, 2004). Still the functional relationship between chromosomal packaging in the nucleus and gene activity is not well defined (Cremer et al., 2004; Dehghani et al., 2005).

1. Positioning of gene loci within the nucleus In vertebrate nuclei chromosomes with low- or high-gene density reside at the nuclear periphery and nuclear interior, respectively (Bolzer et al., 2005; Boyle et al., 2001; Cremer et al., 2003; Croft et al., 1999; Parada and Misteli, 2002; Tanabe et al., 2002). This is nicely illustrated by the observation that two similarly sized human chromosomes (#18 and #19), but with a dramatic difference in gene density (18 < 19) adopt preferred positions in the cell nucleus (Fig. 5.4B). Chromosome 19, which has also a higher level of histone acetylation, has a more internal position in the nucleus whereas chromosome 18 occurs mostly at the periphery (Croft et al., 1999). This gene density-related position of chromosomes #18 and #19 is highly conserved during evolution irrespective of major chromosomal rearrangements (Cremer et al., 2003). In general, condensed “inactive” domains tend to cluster preferentially at the nuclear periphery, whereas less condensed “active” domains locate more often at the interior region. Apart from a general “interior versus peripheral” distribution, there are also chromatin loci that adapt a preferred position with respect to other chromatin structures depending on their state of gene activity (Baxter et al., 2002; Carmo-Fonseca, 2002). Fisher and coworkers showed a dynamic repositioning of certain genes in mouse B and T cells depending on their transcriptional status during cell differentiation. The transcriptionally inactive genes are preferentially localized near pericentromeric heterochromatin, while the active genes were located away from these domains. (Baxter et al., 2002; Brown et al., 1997, 2001; Carmo-Fonseca, 2002). In conclusion, transcriptionally active or silenced gene regions as well as gene rich or poor regions tend to associate with a specific nuclear position. In addition, the association of genes or gene loci with nuclear protein-compartments may also contribute to gene functionality. Several such regulatory compartments can be identified within the interphase nucleus, such as the nucleolus, promyelocytic leukemia bodies (PML), Cajal bodies, SC-35 rich speckles, and various other nuclear domains (Spector, 2001). The function

176

Verschure et al.

Figure 5.4. Active chromatin loops out of compact chromatin regions. Gene activity is related to the location of the gene locus with respect to more compact chromatin structures. (A) The extrusion of a gene outside of compact chromatin is associated with gene activity. These active chromatin structures are suggested to present fine chromatin loops extending away from more condensed chromatin domains. This is exemplified by the “looping out” of the highly expressed IGF2 gene (small arrow) from the painted arm of chromosome 11 (large arrow). Three-dimensional images from interphase primary fibroblast nuclei in which the IGF2 gene is FISH labeled (pointed by small arrows) and the short arm of chromosome 11 is FISH labeled (pointed by large arrows). Bar in (A) represents 4 mm. The figure is kindly provided by Dr. W. A. Bickmore, MRC Human Genetics Unit, Edinburg. (B) Gene density-related radial dependence of chromosomal positioning. Chromosomes with low- or high-gene density are described to reside at the

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

177

of nuclear-protein domains is in many cases not completely understood (Spector, 2001). Their association with specific gene loci is suggested to influence gene activity. For instance, histones and U2 small nuclear RNA genes have been localized proximal to Cajal bodies (Gall, 2001), while PML bodies occur associated with the MHC locus (Shiels et al., 2001; Wang et al., 2004a,b). Furthermore, multiple specific active genes or chromosome segments cluster with SC-35 speckles (Shopland et al., 2003). The nucleolus, the place where ribosomal RNA transcription and processing occurs, is an example of clustering of highly active regions from several chromosomes (Lewis and Tollervey, 2000). Defined nuclear regions may provide a particular gene or gene locus with a high concentration of factors required for gene activity. The interaction of the chromatin with nuclear structures might create anchorpoints for the chromatin, that is, a so-called “matrix” (Ching et al., 2005). Therefore, highly transcribed or silenced loci take a particular position inside the cell nucleus. Still the mechanisms underlying gene control related to nuclear positioning remain to be elucidated. Of interest, the natural ZFP, KRAZ1, and KRAZ2 factors, are found predominantly at defined nuclear chromatin compartments, namely at pericentromeric heterochromatin. This positioning is dependent on the presence of HDAC (the enzyme that downregulates histone acetylation) and is related to the ability of the KRAZ proteins to silence a reporter gene. Namely, HDAC inhibitors abolish both the preferred localization as well as the silencing capacity of KRAZ proteins (Matsuda et al., 2001), (Section I.C for more details). These observations illustrate that the epigenetic environment in the cell nucleus may be linked to the effect of engineered ZFP-TFs.

D. Human diseases and epigenetic gene control mechanisms Various human diseases are assumed to be the result of genomic rearrangements that change epigenetic gene regulation. In addition, certain diseases result from a defined gene mutation that has been shown to affect global epigenetic profiles. In Table 5.1 we highlight some examples of human epigenetically linked diseases. nuclear periphery and nuclear interior, respectively. This is illustrated by the preferred positions of chromosome 18 at the nuclear periphery (left) and 19 at the nuclear interior (right). Of these similarly DNA molecule sized chromosomes, chromosome 19 has the highest observed ratio of gene-based marker assignments of any human autosome, and it is abundantly hyperacetylated at histone H4, whereas chromosome 18 has far fewer gene assignments than expected for its size and it contains only little hyperacetylated histone H4. Two-dimensional images from interphase primary fibroblast nuclei in which two copies of chromosomes 18 (left panel) and 19 (right panel) are painted with FISH (arrow). Bar in AB represents 4 mm. The figure is kindly provided by Dr. W.A. Bickmore, MRC Human Genetics Unit, Edinburgh, Scotland.

Verschure et al.

178

Table 5.1. Overview of Diseases Linked to Epigenetic Dysregulation Disease

Cause

Epigenetic change

Reference

Rett syndrome

Mutation in MeCP2

Fragile X syndrome

CCG expansion 50 untranslated region of FMR1 gene

(Amir et al., 1999; Klose and Bird, 2003) (Harikrishnan et al., 2005; Oostra and Willemsen, 2002; Sutcliffe et al., 1992)

Promyelocytic leukemia

Fusion PML-RAR by translocation between chromosome 15 and 17 Mutation in hemoglobin

Overall changes in DNA methylation FMR1 gene DNA methylation Role for SWI/SNF chromatin remodeling complexes Overall changes in histone acetylation PML bodies dispersed in the nucleus Overall changes in histone acetylation and DNA methylation, RNA involved DNA methylation of imprinted genes altered

(Tufarelli et al., 2003)

Global genomic DNA hypomethylation In tumor cells genes that are silenced by promoter hypermethylation Changes in histone acetylation patterns

(Bachman et al., 2003; Di Croce et al., 2002; Esteller, 2005; Esteller et al., 2001; Feinberg and Vogelstein, 1983; Goelz et al., 1985; Herman and Baylin, 2003; Kondo et al., 2003; Lund and van Lohuizen, 2004). (Melki, 1997; Nicole et al., 2002; Ogg and Lamond, 2002) (Burke and Stewart, 2002; Gruenbaum et al., 2005; MattoutDrubezki and Gruenbaum, 2003)

A-thalassemia (sickle cell anemia)

Imprinting diseases: Prader-Willi syndrome Angelman syndrome BeckwithWiedemann syndrome Cancer

Imprinting defects

Spinal muscular atrophies

Mutation in SMN1

Cajal bodies are dyslocalized

Laminopathies: Hutchinson– Gillford progeria syndrome

Mutation in type A lamin

Nuclear envelope disturbed

(Grande et al., 1996; Grignani et al., 1998; Koken et al., 1994; Zhong et al., 1999).

(Goldstone, 2004; Maher and Reik, 2000; Nicholls et al., 1998)

(Continues)

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

179

Table 5.1. (Continued) Disease

Cause

ICF syndrome

Mutation in Dnmt3b

Polyglutamine diseases: Huntington’s disease

Expansion of DNA triplet in coding region of gene coding for polyglutamine protein

SCA-1, SCA-2, SCA-3, SCA-6, SCA-7 DRPLA Kennedy’s disease

Epigenetic change Hypomethylation of defined chromatin regions HP1 colocalizing with decondensed chromatin domains Polyglutamine protein aggregates and location of aggregates in the nucleus Altered histone acetylation patterns

Reference (Luciani et al., 2005)

(Cha, 2000; Chastain and den, 1998; Lin et al., 2000; Orr, 2001; Yamamoto et al., 2000; Zoghbi and Orr, 2000)

Abbreviations: SCA—spinocerebellar ataxia; DRPLA—dentatorubral pallidoluysian atrophy; ICF—immunodeficiency centromeric instability and facial syndrome; Dnmt3b—de novo DNA methyltransferase; MeCP2—methyl-CpG–binding protein 2; SMN—survival of motor neuron; PML-RARa—promyelocytic leukemia-retinoic acid receptor a; Cajal—nuclear body.

A clear example of regulation via epigenetic modifications is the transmission of imprinted signals during embryonic development. Imprinting describes a change in expression of a gene that occurs during passage through the sperm or egg with the result that the paternal and maternal alleles have different properties in the very early embryo. The specific pattern of DNA methylation in germ cells is responsible for the phenomenon of imprinting. Patterns of DNA methylation are reset during gamete formation. For example, in the H19 gene locus, CpG dinucleotides 6 kilobase pairs upstream and 0.6 downstream of the gene are heavily methylated on the paternal allele and form chromatin that is inaccessible for DNAse I enzymes, compared with the expressed nonmethylated maternal allele. Aberrations in expression of imprinted genes are involved in diseases, including Prader-Willi syndrome, Angelman syndrome, and BeckwithWiedemann syndrome (Goldstone, 2004; Maher and Reik, 2000; Nicholls et al., 1998). In these conditions an abnormal phenotype is established as a result of the absence of the paternal or maternal copy of an imprinted gene, with the silent imprinted gene being unable to compensate for the loss of the active gene, or because of deregulation of an imprinted gene, leading to overexpression. Epigenetic gene control has become an increasingly important aspect of cancer biology (Lund and van Lohuizen, 2004). Cancer epigenetics has mainly focused on aspects of aberrant DNA methylation patterns i.e., global genomic

180

Verschure et al.

hypomethylation in tumors (Feinberg and Vogelstein, 1983; Goelz et al., 1985), identifications of genes that are transcriptionally silenced in cancer cells by promoter hypermethylation of CpG islands (Bachman et al., 2003; Di Croce et al., 2002; Esteller et al., 2001; Herman and Baylin, 2003; Kondo et al., 2003), and the translational use of drugs that act as DNA-demethylating agents (Esteller, 2005). Less attention has been given to histone modifications in cancer cells. However, inhibitors of the enzyme that downregulates histone acetylation, HDAC, are already used in clinical trials and seem to work synergistically with DNA-demethylating agents (Belinsky et al., 2003; Cameron et al., 1999; Pandolfi, 2001; Taddei et al., 2005). In a study by Esteller and coworkers (Fraga et al., 2005), it was demonstrated that histone modification patterns are globally changed in human tumor cells. Their data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells. Of interest, a recent paper demonstrated that human chromatin remodeling factor Brahma (Brm), a catalytic component of the SWI-SNF complex, associates with MeCP2 in vivo and is functionally linked with gene repression (Harikrishnan et al., 2005). The authors showed that Brm and MeCP2 assembly on chromatin occurs on methylated genes related to cancer and on the FMR1 gene in Fragile-X syndrome. This study demonstrates a new role for SWI-SNF chromatin remodeling complexes in gene repression via MeCP2. This finding extends the mechanistic link between DNA methylation, chromatin remodeling activities, and gene repression. Many diseases are related to an altered distribution of nuclear bodies or nuclear proteins. The changes in structure and/or localization of nuclear compartments may result in cellular degeneration, death, deregulation of proliferation, differentiation, and apoptosis thereby contributing to malignant transformation and degenerative diseases. Several examples exist showing a connection between nuclear compartmentalization and changes in cell functionality (Zimber et al., 2004). A well-studied disease related to epigenetic misregulation and altered distribution of a nuclear domain is the ICF syndrome. It involves a rare autosomal-recessive disorder caused by mutation of the gene encoding a de novo DNA methyltransferase DNMT3b. ICF syndrome patients display immunodeficiency, facial anomalies, mental retardation, and developmental delay. The chromosomal abnormalities essentially involve constitutive hypomethylation of satellite 2 DNA mostly located at the juxtacentromeric heterochromatin of chromosomes 1 and 16. This disease demonstrates that hypomethylation of satellite DNA can induce alterations in the structure of heterochromatin. Luciani et al. (2005) analyzed the distribution of HP1 proteins (which are essential components of hetereochromatin and colocalize with condensed chromatin domains) in the lymphoblasts of ICF patients. The authors investigated

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

181

the effect of DNA hypomethylation on hetereochromatin organization in ICF. They observed that during a certain phase of the cell cycle HP1 proteins colocalize with undercondensed 1q or 16q heterochromatin, whereas nuclear bodies such as PML bodies form a large body that colocalizes with the HP1 signal. This is the first description of the relation between hypomethylation and the localization of HP1 proteins in the ICF syndrome. Another example to demonstrate the connection between nuclear organization, genome function and disease are changes in nuclear organization and gene activity observed in neurodegenerative disorders where unstable repeats are translated into an expanded polyglutamine tract. Myotonic dystrophy, spinocerebellar ataxia, and Huntington’s disease are commonly referred to as “polyglutamine diseases” (Orr, 2001; Zoghbi and Orr, 2000). At the protein level the expanded polyglutamine proteins tend to aggregate and form inclusion bodies that are frequently located in the nucleus of affected cells. It is still controversial whether the nuclear aggregations act by sequestering transcription factors in the nucleus, or rather have a protective role in restraining the availability of toxic polypeptides to interfere with transcriptional regulators (Yamamoto et al., 2000). It is suggested that an abnormal interaction between polyglutamine tracks and the transcriptional machinery enhances the disease. Furthermore, the expanded tracts were shown to bind preferentially to specific transcriptional regulators and inhibit the function of others (Cha, 2000; Lin et al., 2000). The idea is that expanded repeats may form secondary structures that confer genetic instability and most likely alter the local chromatin configuration, leading to changes in gene activity (Chastain and Sinden, 1998). Many diseases have an epigenetic component, which is up to now not underscored. There are probably many surprises ahead. It is interesting to note that both muscular-neurodegenerative diseases and cancers are associated with abnormalities and functional disorganization in nuclear subcompartments. Both classes of diseases display opposite phenomena, namely excessive cell death in muscular-neurodegeneration and excessive cell proliferation and survival in neoplastic diseases. We expect that in coming years, such epigenetic aspects of human diseases will obtain increasing attention. Elucidating the various levels of epigenetic gene regulation in human diseases is an exciting challenge that will eventually lead to a better understanding of development of such diseases and will boost new directions of therapeutic strategies. In the following section, we focus on the use of ZF-TF to modulate epigenetic gene regulation. At the moment there is only scarce information about the effect of ZF-TFs at the epigenetic level. Most of the knowledge has been gained at the nucleosomal level. However, we should keep all the epigenetic gene-control levels (involving control at nucleosomal, large-scale chromatin, and nuclear level) in mind when scrutinizing ZF-TF effects on epigenetic gene regulation.

182

Verschure et al.

II. HOW DO ZF-TFS MODULATE EPIGENETIC GENE REGULATION? Gene expression is regulated both via the DNA sequence by the composition of the promoter and enhancer sequences and via epigenetic control levels. For the purpose of modulating gene expression via epigenetic gene control, the nucleosomal level provides the best understood molecular mechanisms. Several approaches are being taken to modify histone modifications with therapeutic intentions, including a general inhibition of HDACs (the enzymes that remove acetyl groups from histones, thereby upregulating histone acetylation). The use of engineered ZF-TF is an emerging technology to modify the expression of a specific gene. A dozen genes have been up- or downregulated (as reviewed in the accompanying Chapter 4 by Visser et al.). ZF-TFs consist of an engineered ZFP that recognizes DNA in a sequence-specific manner, fused to an effector domain. The modular character of transcription factors allows natural transcriptional effector domains to be stitched to any engineered ZFP (Beerli et al., 2000; Liu et al., 2001). Among the domains most often exploited are the activator domain VP16 and repression domain Kruppel associated box (KRAB). Depending on the effector domain fused to the DNA binding module, ZF-TFs can thus either silence or activate gene expression. Here, we provide an overview of the effector domains used (Table 5.2) and discuss how these domains interact with epigenetic parameters.

A. Activation effector domains Several activation domains have been fused to zinc-finger DNA-binding domains to upregulate the expression of an endogenous gene. An overview is given in Table 5.2. One of the most used and best-studied activating domains is the highly acidic portion of the herpes simplex virus transactivating protein VP16 (Sadowski et al., 1988) and V64, which consists of four minimal VP16 activation subunits (Beerli et al., 1998). VP16 has been shown to interact with a number of transcription factors, including TFIIB, TFIIH, TATA-binding protein (TBP), TBP-associated factors (TAFs), host cell factor (HCF), and Oct-1(Herrera and Triezenberg, 2004), suggesting that VP16 activates transcription by inducing the assembly of an RNA polymerase-II (RNA Pol II) preinitiation complex. It is hypothesized that for gene activation to occur not only the RNA pol II complex is required, but that also chromatin needs to obtain a decondensed, open chromatin structure (Nye et al., 2002). VP16 appears to be able to realize both these conditions, as it interacts directly or indirectly with chromatin-modifying enzymes, such as proteins that exhibit HAT activity, including CBP, p300, the hGCN5 complex, and components of the SWI/SNF chromatin remodeling complex (Herrera and Triezenberg, 2004; Memedula and Belmont, 2003). When a VP16 lac repressor-GFP-tagged fusion protein is targeted to an amplified chromosome region containing multiple lac-operator repeats, the amount of

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

183

Table 5.2. Overview of ZFP Fused Gene Expression Modulating Effector Domains Effector domain VP16

VP64

p65 domain from NF-B transcription factor

C-terminal domain or 6–11 repeats of FDTDL motive from -Catenin KRAB KRAB-A box

Interaction

Action

Effect

Transcription factors: TFIIB, TFIIH, TBP, TAFs, HCF, Oct-1 Histone acetyltransferases: CBP, p300, hGCN5 complex Chromatin remodeling factors: SWI/SNF complex Transcription factors: TFIIB, TBP

Assembly polymerase II preinitiation complex Histone acetylation Chromatin decondensation

Efficient upregulation of gene expression, including permissive and silent imprinted genes Partially TATA-box dependent Efficient up regulation of plasmid reporter gene Efficient upregulation of gene expression

Histone acetyltransferases: CBP, p300 Histone acetyltransferases: CBP, p300 (HAT activity not required)

Transcription factors: TATA-dependent basal transcription machinery Interaction via KAP-1 with HP1, HDACs and SETDB1

TR and vErbA

Transcription factors: TFIIB, several corepressors including SMRT and NcoR Histone methyltransferases: SUV39H1 (hormone independently) Chromatin remodeling factor: NURD

Assembly polymerase II preinitiation complex Histone acetylation

Interference with assembly of polymnerase II preinitiation complex Creation of heterochromatintype environment Sequestration to heterochromatin nuclear compartment (speculative) Preventing formation of preintiation complex Histone deacetylation and methylation

11 repeats of FDTDL motive upregulate VEGF-A gene expression more potent then single VP16 Efficient downregulation of gene expression, both from endogenous chromatin and reporter plasmids. (the latter most likely via interference with the TATA box complex) Downregulation of gene expression (TR in absence of hormone). vErbA No downregulation of gene expression of naked plasmid reporter gene

(Continues)

Verschure et al.

184 Table 5.2. (Continued) Effector domain SID

HMT domain of G9A and SUV39H1

SRDX from plant transcription factor SUPERMAN

Interaction

Action

Transcription factors: transcriptional corepressor mSIN3

Histone methylation

Effect Downregulation of gene expression induced by transcriptional activators Downregulation of gene expression No downregulation of gene expresion in naked reporter plasmid Downregulation of gene expression of hVEGF-A only during hypoxia

Abbreviations: VP64—4 minimal acidic activation domains of VP16; VP16—from herpes simplex virus transactivating protein; TFIIH and TFIIB—transcription factors; TBP—TATAbinding protein; TAF—TBP-associated factors; HCF—host cell factor; Oct-1—transcription factor; NF-B—transcription factor; -catenin—transcription factor; SRDX—12 amino acids stretch (LDLDLELRLGFA) based on the EAR transcriptional repression motif derived from SUPERMAN; SUPERMAN—transcription factor in plants; mSIN3—transcriptional corepressor; SID—mSin3 interaction domain from Mad transcription factor; vErbA—virally derived mutated version of chicken TR, which is hormone independent; TR—thyroid hormone receptor; KRAB—from human Kruppel-associated box, domain of the KOX-1 protein; KAP-1—protein involved in heterochromatinization; HP1—heterochromatin protein, protein involved in heterochromatinization; CBP—CREB-binding protein, protein with acetyltransferase activity; p300—protein with acetyltansferase activity; HAT—histone acetyl transferase (upregulation histone acetylation); HDAC—histone deacetylase (downregulation of histone acetylation; SETDB1—histone methyltransferase; SUV39H1—histone methyltransferase; G9A—histone methyltransferase; SMRT— silencing mediator for retinoic acid receptor and TR; NcoR—nuclear receptor corepressor; NURD—nucleosome remodeling and histone deacetylation.

histone H3 and H4 acetylation of that region increases, and the chromatin structure of the amplified chromosome region decondenses (Memedula and Belmont, 2003). However, the observed chromatin decondensation is not sufficient for transcription to occur (Carpenter et al., 2005; Nye et al., 2002). It is not known whether the targeting of a single VP16 via a ZFP is sufficient to induce decondensation of the chromatin structure of the targeted gene. The efficiency with which a targeted ZFP-VP16 activates the targeted gene depends on its proximity to the TATA box (Stege et al., 2002). It is also observed that ZFPVP16 targeting to the TATA box-free promoter of the ErbB-2 gene in mouse fibroblasts could increase expression only moderately, compared with targeting

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

185

the same ZFP in human HeLa cells, in which the ErbB-2 gene promoter does contain a TATA box (Beerli et al., 2000). Most likely, ZFP-VP16 and ZFPVP64 can be used efficiently to switch a gene from off to on when targeted close to the TATA box. However, when no TATA box is present in the promoter region, VP16 may be able to enhance ongoing transcription by creating a chromatin environment that is more supportive for transcription but probably does not initiate transcription by itself. Another activation domain that has been coupled to ZF-TF to activate endogenous transcription is the NF-B transcription factor p65 domain (Bae et al., 2005; Jouvenot et al., 2003; Liu et al., 2001). The p65 domain (amino acids 288–551) contains abundant hydrophobic, and serine amino acids, which are essential for its transactivation activities. p65 has been found to interact with coactivators CBP/p300 (Ashburner et al., 2001) as well as with the general transcription factors TATA-box–binding protein (TBP) and TFIIB (Schmitz et al., 1995). The C-terminal domain of -catenin has also been used as activation effector domain fused to ZF-TF (Tachikawa et al., 2004). Also for this domain, an interaction with CBP/p300 has been described, although the intrinsic histone acetyl transferase activity of p300 is not required for this effect (Hecht et al., 2000).

B. Comparison of the effect between different activation domains Although some studies have investigated several activation domains, no thorough comparison among the effects of different domains has been studied. Jouvenot et al. (2003) compared the effect on gene regulation of targeting VP16 and p65 when fused to the same ZFP. For the IGF2 gene, both effector domains achieved about a 40-fold activation. For the H19 gene, where the ZF-TF was targeted close to the start site of the gene, VP16 enhanced the H19 expression about 40, while p65 reached a 130-enhancement. In another study, Liu et al. (2001), showed that for most ZFPs there was little difference in the potency between VP16 and p65, with p65 being maximally 3-more potent than VP16. Tachikawa et al. (2004) compared the potency of several activation domains, fused to the same ZFP, to induce VEGF-A gene expression. They found that VP16 was much more potent then -catenin. Interestingly, they observed that the use of a single copy of the FDTDL motif from -catenin did not have any effect, but if the copy number of this motif was increased, it progressively became a very powerful activator. Six repeats were more effective than -catenin itself, and 11 repeats of the motif exceeded the effect of VP16 (Tachikawa et al., 2004). The partial dependency of VP16 on the TATA box was not noted for the other activation domains. Strikingly, when both VP16 and p65 activation

186

Verschure et al.

domains where targeted to neighboring sites in the VGEF-A promoter, Liu et al. (2001) observed dramatically enhanced gene expression, far more then each effector domain targeted separately. The enhanced expression was not observed when the same effector domain was targeted twice to neighboring sites, suggesting that VP16 and p65 have a slightly different mechanism of action and that the simultaneous presence of these domains on a chromatin region results in a synergistic effect. This indicates that when choosing the effector domain for a ZF-TF, one should take into account the promoter structure of the targeted gene. If no TATA box is present, VP16 cannot be expected to initiate gene expression, and one of the other effector domains would be preferred. To boost expression levels further, one may consider targeting two ZFP to neighboring sites, with two synergistic effector domains, such as VP16 and p65.

C. Repression effector domains A variety of repression domains derived from natural transcription factors have been fused to ZFPs to silence a defined gene (Table 5.2). Similar to the activation domains, most repression domains are expected to act, at least in part, by recruiting chromatin-remodeling enzymes. Recruitment of such enzymes, for example, histone deacetylases that downregulate histone acetylation or histone methyltransferases that upregulate histone methylation, are involved in the induction of gene silencing and the formation of facultative heterochromatin (Thiel et al., 2004). One of the most commonly used repression domains fused to engineered transcription factors is the human Kruppel-associated box (KRAB) domain of the KOX-1 protein. Within the large family of zinc-finger transcription factors, most factors in vertebrates contain KRAB domains as effectors. The domain is divided in A and B boxes and is highly conserved among eukaryotes (Margolin et al., 1994). The A box is involved in binding corepressors; the B box enhances the repression, but its exact mechanism is still unknown. Not all natural KRAB-containing ZFP-TFs contain the B box. A minimal domain of 45 amino acids in the KRAB-A box is sufficient for gene repression (Margolin et al., 1994). Different TFs have been constructed utilizing either the complete KRAB repression domain or only the KRAB-A box to achieve gene repression (see accompanying Chapter 4 by Visser et al.). The KRAB domain has been shown to function through direct interaction with the TATA-dependent basal transcription machinery. Although this machinery is used both by TATA box-containing promoters as well as by Inr-promoters, which contain a pyrimidine-rich initiation element, KRAB was found to strongly repress TATA-box-dependent promoters and only to weakly repress Inr-promoters (Pengue and Lania, 1996). This differential activity has also been observed for other transcriptional

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

187

modulators (p53, VP16) and might represent an important aspect for the design of ZF-TFs. Kruppel-associated box also mediates transcriptional repression via an epigenetic regulated mechanism. After binding of the KRAB domain a facultative heterochromatin environment is formed on the target promoter of the gene, and expression of the gene is silenced (Urrutia, 2003). This is achieved by the recruitment of KRAB by Kap1, which is suggested to form a scaffold containing the heterochromatin-associated protein HP1, HDACs, and the histone methyltransferase SETDB1 (Schultz et al., 2002). Similarly, the direct targeting of HP1 as a HP1-lac repressor-GPF–tagged fusion protein to a megabase pair-sized amplified chromosome region consisting of lac operator sequences, resulted in recruitment of histone methyltransferase SETDB1 and increased levels of trimethylation of histone H3 at lysine 9. This targeting of many HP1 fusion proteins was shown to condense the chromatin region (Brink et al., 2006; Verschure et al., 2005) (see also Section I.B.2). It is unknown whether the targeting of a single KRAB domain by a ZFP is also able to initiate chromatin condensation, as described for lac-repressor targeting to a stretch of lac-operator binding sites. Matsuda et al. (2001) showed that the natural KRAB ZFPs, KRAZ1, and KRAZ2, are sequestered to pericentromeric heterochromatin by their interaction with KAP-1 and HP1. The interaction with both KAP-1 and HP1 were shown to be required for both the sequestering and silencing of a reporter plasmid. Both the silencing and the sequestering to pericentromeric regions, were dependent on histone acetylation levels, as treatment with the HDAC inhibitor TSA caused a partial redistribution of the KRAZ proteins away from the heterochromatin and partly reversed the silenced gene expression. Neither silencing, nor the sequestering to pericentromeric heterochromatin of the KRAZ ZFPs, could be counter acted by cotargeting VP16 via the expression of a KAP-1-VP16 fusion protein. This might indicate that the pericentromeric region is a transcriptionally inert compartment where even strong transactivation via VP16 is severely suppressed (Matsuda et al., 2001). These observations bring forward an interesting thought that KRABZFP may act via three pathways—first, by interfering with the TATA box complex assembly; second, by recruiting enzymes to change the local epigenetic environment, and finally, by relocating the targeted gene to a nuclear environment that involves gene silencing. This is supported by a study of Ayyanathan et al. (2003), demonstrating that the local recruitment of KRAB to a reporter gene (two to five copies integrated in a single locus), resulted not only in silencing of the reporter gene, but also in a local accumulation of KAP-1 and HP1, DNA methylation, a compact nucleosome structure, and relocation of the gene to heterochromatin compartments in the nucleus. Transient targeting of the KRAB repressor domain to the reporter gene resulted in a mitotically inheritable silencing of the reporter gene in a subset of the cells.

188

Verschure et al.

Other promising naturally occurring repression domains used in ZF-TF research are the inducible ligand dependent thyroid hormone receptor (TR) (Snowden et al., 2003), its viral relative vErbA, which is a constitutive repressor (Jouvenot et al., 2003; Snowden et al., 2003) and the mSin3 interaction domain (SID) (Magnenat et al., 2004). SID is a 35 amino acids domain located in the N-terminus of the transcription factor Mad. SID has been shown to recruit the transcriptional corepressor mSIN3. When fused to a DNA binding domain, SID strongly repressed gene expression induced by transcriptional activators (Ayer et al., 2005). The thyroid hormone receptor is extremely useful for therapeutic gene silencing, as the repression is hormone-dependent and can be switched on or off by providing hormone. In the absence of hormone, TR interacts with TFIIB and several corepressors, including silencing mediator for retinoic acid receptor and TR (SMRT), and nuclear receptor corepressor (NcoR), thereby preventing formation of the preintiation complex (PIC) that is necessary for transcription. Moreover, in the absence of hormone, these corepressors have been shown to recruit HDACs. When ligand is present, conformational changes that occur when the ligand binds to the receptor results in dissociation of almost all corepressors and relief of repression (Eckey et al., 2003). In addition, TR recruits histone methyltransferases (e.g., SUV39H1 interacts with TR in a hormone independent manner) as well as the chromatin remodeling factor nucleosome remodeling and histone deacetylation (NURD). vErbA is the virally derived mutated version of chicken TR and contains nine point mutations resulting in a low affinity for the thyroid hormone. Its repression activities are therefore hormone-independent and believed to be less effective compared with TR itself. The inefficiency has been ascribed to the low affinity for the transcription factor TFIIB (Urnov et al., 2000). However, a direct comparison in which TR and vErbA were fused to the same ZFP showed that both repressors were equally potent (Snowden et al., 2002). If an induction system is not required, vErbA may be preferred, as regular culture medium contains sufficient trace amounts of hormone to diminish the gene-silencing capacity of TR. However, as vERbA has oncogenic potential this might not be the most preferred repression domain to be exploited for ZF-TF designed for therapeutic treatment. ZFP-vERba was shown to recruit HDAC, resulting in deacetylation of histone H3 and H4 and gene inactivation (Urnov et al., 2000), whereas chemical inhibition of HDAC almost completely abolished the gene silencing (Snowden et al., 2003). Interestingly, both TR or vErbA based ZF-TF were able to repress a targeted gene only in the context of endogenous chromatin, while the same ZF-TF was unable to repress a reporter gene containing the identical natural promoter sequences but introduced as naked DNA plasmids (Snowden et al., 2003). This illustrates that epigenetic gene regulatory factors,

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

189

such as histone modifications that are induced by these domains, are an important mechanism of gene silencing. The direct targeting of the catalytic subunits of histone methyltransfereases G9A and SUV39H1, via a ZFP to the VEGF-A gene, was shown to be sufficient to induce gene silencing to similar levels as targeting of vErbA (Snowden et al., 2002). Interestingly, the fusion of full-length SUV39H1 ZFP was not able to repress VEGF-A gene expression. The authors speculate that this may be due to the interaction of the full-length SUV39H1 protein with HP-1. HP1 is believed to sequester the ZF-TF to HP1-rich heterochromatin domains and thereby to compete with DNA binding of the ZF-TF at the VEGF-A gene (Snowden et al., 2002). The catalytic subunits of SUV39H1 and G9A induced histone methylation not only at the ZFP-targeted site, but also at least 900 base pairs away (which corresponds to roughly five nucleosomes). This “spreading” effect could be caused by the natural effect of the histone methyltransferase SUV39H1 (i.e., recruitment of endogenous HP1 and induction of enhanced trimethylation of histone H3 at lysine 9), which in turn interacts with endogenous SUV39H1. At present, the reach of such a spreading mechanism is unknown. The targeting of KRAB to an active reporter gene resulted in a local accumulation of KAP-1 and HP1, limited to the target site, the promoter, and the transcription initiation site, while the 30 end of the reporter gene showed no signal for KAP-1 or HP1 (Ayyanathan et al., 2003). Even so, this spreading effect should be taken into account when designing repression factors for closely spaced regulatory elements for two neighboring genes. Specialized boundary elements have been identified to border chromatin domains with distinct functionality (transcriptional inactive versus active) as described in more detail in Section I.B.1. If no such boundary elements are present to prevent spreading, it is conceivable that a neighboring gene may be affected as side effect. Alternatively, local spreading of a functional chromatin state may also be caused by physical interactions of the targeted catalytic domain with the targeted histones. The effect of an interaction between two nucleosomes in in vivo folded chromatin is illustrated by the targeting of ZFP fused to a de novo DNA cytosine methyltransferase (DAM). In this regard, Carvin et al. (2003) targeted DAM in yeast cells, which naturally do not have methylated DNA. The authors showed that apart from the targeted site, increased methylation was also found at some distance from the targeted site (at 180 and 360 base pairs), suggesting that indeed the enzyme activity spreads by 1 or 2 nucleosomes. This distant methylation is proposed to result from a physical interaction of the enzyme with DNA wrapped around neighboring nucleosomes. Although theoretically targeting DAM activity can be used to epigenetically silence genes, the observed background levels of DAM activity in this study were too high to allow induction of gene-specific silencing (Carvin et al., 2003).

190

Verschure et al.

Gene-repression mechanisms appear to be well conserved, as also plant derived transcriptional repressors have been shown to function in mammalian cells—SRDX is a 12-amino acid stretch (LDLDLELRLGFA), which is based on the EAR transcriptional repression motif derived from the plant transcription factor SUPERMAN (Hiratsu et al., 2003). Curiously, this repressor has been shown to inhibit human VEGF-A production only under hypoxia conditions (Tachikawa et al., 2004). If further research substantiates this feature, the inducible character may be useful in a therapeutic setting.

D. Comparison of the effect between different repression domains Most repressors studied are similarly potent in effecting gene repression, although the actual repression achieved depends on the exact combination of ZFP, effector domain, and even cell type, as is illustrated by Magnenat et al. (2004). The authors compared four zinc fingers, initially selected for induction of the ICAM-1 gene by VP16, and fused to repression domains KRAB and SID. One ZFP fused to KRAB repressed ICAM levels almost completely, but not when fused to SID. The other ZFP-TFs repressed the ICAM gene expression to some extent; some repressed better with SID, others with KRAB. Since no causative reason could be determined for the difference between particular ZFPTFs to repress gene activity, experimental optimization is required. Tachikawa et al. (2004) compared the effect of KRAB, SID, and SRDX. The VEGF-A gene was repressed to about 20% of normal levels by either KRAB or SID. Curiously, the SRDX domain had no effect on VEGF-A gene expression in normal cells, but under hypoxic conditions it did repress expression to the same levels as KRAB and SID. Further studies are required to confirm the inducible character of the SRDX domain. The repression of vErbA was shown to be similar to that of the catalytic units of the histone methyltransferases G9A and SUV39H1 (Snowden et al., 2002). These few studies indicate that there is not a universal superior repression domain, and that KRAB, SID, vErbA, and histone methyltransferases all have a similar efficiency in decreasing gene activity. Potentially, the epigenetic microenvironment may create some differences in the effects of particular effector domains in context of a certain chromatin structure. It is therefore interesting that a cooperative action of cotargeted effector domains can be observed. Most natural TFs act by interacting with a variety of other proteins, such as histone-modifying enzymes, DNA methyltransferases, HP1, and other epigenetic factors. Together, these enzymes reset the local chromatin structure. Snowden et al. (2002) showed that by targeting two complementary effector domains (the catalytic unit of the histone methyltransferase G9A, and the transcriptional repressor v-ErbA, which attracts histone deacetylases) to neighboring sites, the repression was further enhanced, compared with targeting each factor alone.

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

191

This indicates that selecting and cotargeting more then one complementary factor can increase the effect of the ZF-TFs dramatically.

III. EPIGENETIC ASPECTS TO CONSIDER FOR ZF-TF APPROACHES A. Functional chromatin status versus ZF-TF control Having outlined the factors that are known to influence the effect of an effector domain fused to a ZFP we now consider the following three situations.

1. To induce silencing of a gene that is transcriptionally active This kind of modulation is considered to be efficiently achievable by ZF-TF. The chromatin of an actively expressing gene is considered to be “open” and accessible for (engineered) TFs. The ZF-TF is thus likely to be able to bind the gene and modulate its expression. Of importance, once the ZF-TF has bound the gene, the effector domain should be sufficiently strong and versatile to recruit the enzymes required to change the gene’s expression and local chromatin environment. The gene modulation might induce heterochromatinization and, speculatively, possibly even relocation of the gene toward a “silencing compartment” in the nucleus (Ayyanathan et al., 2003; Urrutia et al., 2003).

2. To induce upregulation of a gene that shows a basal expression level or is not expressed but which is permissive for expression Conceptionally this kind of modulation is the simplest scenario. Here the chromatin of the gene to be modulated is likely in an open configuration and waiting for the correct signals. The engineered ZF-TF may act as a natural transcription factor. Most likely the modulated genes that have been studied so far (see accompanying Chapter 4 by Visser et al.) fit in this category, as many of them can be both up- and downregulated.

3. To induce activation of a gene that is silenced This kind of modulation can be considered the most challenging scenario. The gene is expected to be located in a heterochromatin-like environment. The transcriptional machinery is not expected to be sterically hindered by the compactness of the chromatin as described in Section I.B.3; however, proteins involved in silencing may obscure the actual binding site of a ZF-TF. Also, when a ZF-TF is able to bind, it needs to attract enzymes that can reset the local

192

Verschure et al.

chromatin environment to be accessible for all factors involved in transcription and possibly relocate the gene toward a transcription-compatible region in the cell nucleus. Several genes have been both up- and downregulated by ZF-TF and fall in scenarios 1 or 2, The most studied gene in this respect is the vascular endothelial growth factor VEGF-A. From a medical point of view, both upand downregulation are of importance—in tumors, downregulation of overexpressed VEGF-A would prevent the formation of new blood vessels that sustain the tumor, while under other circumstances, such as ischemia, activation of VEGF-A is beneficial to stimulate the formation of new vessels. In a series of studies ZFPs were identified and subsequently fused to a variety of effector domains and tested under many conditions. These results revealed that, by targeting either VP16 or p65, the expression levels of the VEGF-A gene could be enhanced even to higher levels than levels induced by the natural stimulus of hypoxia (Liu et al., 2001). To repress expression of VEGF-A gene activity, the v-ErbA repression domain as well as the catalytic units of the histone methyltransferases G9A and SUV39H1, were effectively fused to zinc fingers (Snowden et al., 2002). Even in the tumorgenic cell line U251MG, which expresses high levels of VEGF-A gene product, the fusion of vErbA or TR domains to ZFP reduced VEGF-A’s expression level to that of normal cells (Snowden et al., 2003) (in the tumorgenic cell line about 8 more VEGF-A gene product is produced than the expression in nontumorgenic cells). Similarly the ErbB-2 and ErbB-3 genes were each specifically up- and downregulated by ZFP containing either VP64 or KRAB, respectively (Beerli et al., 2000). However, not all ZFP that are effective in upregulating a gene are able to downregulate the same gene when fused to a repression domain. Magnenat et al. (2004) selected four ZFP-VP64 that could efficiently upregulate the ICAM-1 gene. When the VP64 domain was replaced by the KRAB repression domain, only one of the four ZFP fusions fully repressed ICAM-1 expression. The other ZFPs, fused to KRAB or SID, were not efficient in downregulation. This suggests that the epigenetic microenvironment of the targeted ZF-TFs plays a major role in the final effect. Activation of an imprinted gene that is either expressed solely from the paternally derived chromosome or the maternally derived chromosome illustrates that scenario 3 is also feasible (i.e., to induce activation of a gene that is silenced). Jouvenot et al. (2003) showed that the stably silenced H19 and IGF2 imprinted genes could be efficiently activated by ZF-TF. So far, the three mentioned scenarios have been shown to be feasible: (i) to induce silencing of a gene that is transcriptionally active, (ii) to induce upregulation of a gene that shows a basal expression level, or is not expressed but which is permissive, and (iii) to induce activation of a gene that is silenced. Application of ZF-TFs can be considered to have high potential as powerful tools for gene modification. In the

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

193

ensuing paragraph we will discuss some of the issues that can help in the design of successful ZF-TFs.

B. Target definition It is generally believed that accessibility of chromatin determines the ability of ZF-TFs to reach their target genes. From studies performed at the “naked DNA level” (e.g., in a reporter plasmid), clear differences in ZF-TF efficiency compared with the effect at the chromatin level are at least in part believed to be caused by the (in)ability of a ZF-TF to bind chromatinized DNA. Also in vivo natural DNA-binding factors bind only a subset of their recognition sequences (e.g., of the 1286 perfect recognitions sequences of Gal4p in budding yeast, only 10 are bound by the protein in vivo [Ren et al., 2000]). Winding of the DNA around nucleosomes shields parts of the DNA’s major groove from binding a (engineered) TF (illustratively reviewed in Collingwood et al., 1999). In vitro, DNA-binding proteins are able to bind nucleosomal DNA and thereby shift the nucleosomal position to favor stable binding (Li and Widom, 2004; Li et al., 2005). Such features can play a role in vivo as well, although binding of DNA factors to chromatin in vivo is not yet well understood. One approach to bypass nucleosome shielding is to design ZF-TFs that bind in nuclease-hypersensitive sites. This approach assumes that these sites contain stretches of nucleosomefree chromatin to which the ZF-TFs can bind DNA unhampered by the nucleosome (see accompanying Chapter 4 by Visser et al.). However, as demonstrated by Zhang et al. (2000), DNAse I hypersensitivity of the target site is neither a foolproof guarantee nor an absolute prerequisite for ZF-TF binding and functionality. Even so, these investigators observed that one of their most effective ZF-TF was targeted in the linker DNA next to a positioned nucleosome. At the chromatin level one might intuitively believe that the compact organization of heterochromatin excludes a ZF-TF from reaching its target gene. However, it has been demonstrated that large macromolecules with molecular weights of several hundred kilodaltons can access condensed heterochromatinlike domains (Verschure et al., 2003). Also, studies that analyzed targeting of lac repressor-tagged regulatory proteins to an amplified lac operator-containing region demonstrated that even targeting of optically dense heterochromatin is feasible and results in changes in large-scale chromatin configuration accompanied by changes in histone modification state (Memedula and Belmont, 2003). Notably, the regulatory proteins used in these lac operator–lac repressor studies are in the size range of ZF-TFs. Moreover, Jouvenot et al. (2003) showed that even silenced imprinted alleles could be activated by ZF-TF. These observations indicate that targeted genes within silenced chromatin either remain accessible and are not shielded by heterochromatin-binding proteins, or they can become opened up.

194

Verschure et al.

A second consideration when designing a ZF-TF target site is the choice of the effector domain. For many such domains the exact distance to the transcriptional start site appears not critical. For VP16/VP64, however, the number of base pairs between the ZF-TF target site and the TATA box has a clear influence on the efficiency of activation (Stege et al., 2002).

C. Specificity A ZFP based on 6 zinc fingers can bind a strech of 18 base pairs and can therefore theoretically be designed to target a single, unique sequence in the human genome. This was demonstrated by Tan et al. (2003). They repressed the CHK2 gene by a ZFP-KRAB and observed that, only this single gene was affected out of about 16,000 genes tested by DNA microarray. Apart from the selection of ZFP to bind a single site, the selection of the choice of effector domain may also play a role in the specificity obtained. Repression domains, such as KRAB and histone methyltransferases, were found to result in a spreading effect of the enzyme over the chromatin in cis (Ayyanathan et al., 2003; Snowden et al., 2002). It is therefore theoretically conceivable that ZF-TFs can affect a close-by promoter region of a neighboring gene. A second mechanism by which the effector domain can affect expression levels of neighboring genes is by causing a physical relocation of the targeted gene to another functional region in the nucleus, for example, a pericentromeric heterochromatin region (Ayyanathan et al., 2003). Neighboring genes may be affected by such relocation into a “silencing compartment.” However, dedicated studies are required to evaluate whether a single ZF-TF translocates their targeted gene to such a (silencing) compartment and whether this will affect neighboring genes. A third concern about specificity is the effect of overexpression of the ZF-TF-effector domain. For an enzymatically active effector domain, interaction with random DNA may be sufficient to result in a local modification. For instance, DAMfused to a ZFP was shown to result in high-levels of background DNA methylation (Carvin et al., 2003), likely due to nonspecific interactions of the enzyme with DNA.

D. Efficiency Most natural TFs act by interacting with a variety of regulatory proteins, such as histone methyltransferases, DNA methylates, histone (de)acetylases, HP1, and other epigenetic factors (Table 5.2). Together, these enzymes are thought to reset the local chromatin structure. An “attack on two fronts” may be of particular relevance to activate an epigenetically silenced gene. Complementary effector domains can be cotargeted to neighboring sites to stimulate modulation of the local chromatin environment via ZF-TFs. For instance, when activating

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

195

domains VP16 and p65 (Liu et al., 2001) or silencing domains v-ErbA and a HMT (Snowden et al., 2003) were targeted, indeed, this caused increased activation and repression, respectively. As yet, there is little information available regarding the epigenetic gene control via ZF-TFs at the nuclear and largescale chromatin organization level. We can speculate that one effector domain may be able to pull the target gene into a different nuclear compartment (e.g., pericentromeric hetrochromatin or euchromatin), while a second ZF-TF can then act on the gene itself thereby activating the transcription machinery. So far, ZF-TFs have been applied to target single specific genes. We can imagine also that entire gene-clusters may be affected at once. This could be achieved either by designing ZF-TF to target regulatory sequences found in coregulated genes, or more speculatively, by using effector domains that can spread the epigenetic status over larger chromatin regions. In the latter case, boundary elements form a parameter of large-scale chromatin structure that influence the extent of spreading of an epigenetic status, thereby influencing ZF-TF function. Unfortunately, such boundaries are still ill-defined elements, and their function in different chromatin contexts is not well known.

E. Concluding remarks The effect of ZF-TF is clearly not only dependent on its ability to bind a specific DNA sequence in the test tube, but also depends on epigenetic gene control systems that act at the in vivo chromatin level. Many correlations have been described relating organization of the genome in the nucleus with gene control. However, the molecular mechanisms underlying these correlations are still largely unresolved. At the moment most knowledge of epigenetic control mechanisms are available at the nucleosomal level (such as histone modifications), but even here our understanding is incomplete and still in a state of experimental flux. Interestingly, different gene-activation and -repression domains show different effects on nucleosomes. VP16 and KRAB are able to up- and downregulate, respectively, gene expression of both endogenous genes and naked plasmid DNA. The latter is most likely the result of interactions with a TATA box-dependent complex. In contrast, the repression domain vErbA acts mainly via attracting HDACs, and the repression domains catalytic subunits of histone methyltransferases G9A and SUV39H1 act by efficiently repressing an endogenous gene in its normal chromatin environment, but not a plasmid reporter gene (Snowden et al., 2002). Knowledge of the mode of action of a particular effector domain to be fused to a ZFP for gene modulation can greatly enhance the effectiveness of a designed ZF-TF. Various endogenous genes have been successfully targeted by ZF-TFs, such that the expression of targeted genes is known to be modulated. However, these studies provide only spare information on the

196

Verschure et al.

influence of epigenetic gene regulatory aspects. For instance, these studies report only on epigenetic aspects at the nucleosomal level not taking aspects at higher-order chromatin or nuclear level into account. Local epigenetic gene-regulatory systems that control the nuclear microenvironment play an important role in the mode of action of effector domain tagged ZF-TF. In Section I, we highlighted several aspects of such epigenetic gene control systems that influence the nuclear microenvironment. In Section II, we described several features revealed from analysis of effector domains that have been used (so far) to induce gene modulation via ZF-TF. In the last section we described epigenetic phenomena that influence the efficiency of a ZF-TF. In our opinion ZF-TF themselves will become very useful to study the regulatory effects of epigenetic gene-control systems within the interphase cell nucleus. ZF-TF that act as targetter for a defined gene can be considered as a tool to change the epigenetic status of a particular chromatin region. This novel tool will stimulate the development of additional ways to understand epigenetic gene control systems, as it will allow investigators to manipulate various components of epigenetic systems.

Acknowledgments PJV is supported by The Netherlands Organization for Scientific Research (NWO) (VIDI 2003/ 03921/ALW/016.041.311). AEV is supported by The Netherlands Genomics Initiative (Horizon Breakthrough 050-71-009) and by Philip Morris Incorporated.

REFERENCES Allshire, R. (2002). RNAi and heterochromatin: A hushed-up affair. Science 297, 1818–1819. Amir, R. E., Van den Veyver, I. B., Wan, M., Tran, C. Q., Francke, U., and Zoghbi, H. Y. (1999). Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 23, 185–188. Ashburner, B. P., Westerheide, S. D., and Baldwin, A. S., Jr. (2001). The p65 (RelA) subunit of NFkappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol. Cell. Biol. 21, 7065–7077. Ayer, D. E., Laherty, C. D., Lawrence, Q. A., Armstrong, A. P., and Eisenman, R. N. (2005). Mad proteins contain a dominant transcription repression domain. Mol. Cell. Biol. 16, 5772–5781. Ayyanathan, K., Lechner, M. S., Bell, P., Maul, G. G., Schultz, D. C., Yamada, Y., Tanaka, K., Torigoe, K., and Rauscher, F. J., III. (2003). Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: A mammalian cell culture model of gene variegation. Genes Dev. 17, 1855–1869. Bachman, K. E., Park, B. H., Rhee, I., Rajagopalan, H., Herman, J. G., Baylin, S. B., Kinzler, K. W., and Vogelstein, B. (2003). Histone modifications and silencing prior to DNA methylation of a tumor suppressor gene. Cancer Cell 3, 89–95.

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

197

Bae, K. H., Kwon, Y. D., Shin, H. C., Hwang, M. S., Ryu, E. H., Park, K. S., Yang, H. Y., Lee, D. K., Lee, Y., Park, J., Kwon, H. S., Kim, H. W., et al. (2005). Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat. Biotechnol. 21, 275–280. Baxter, J., Merkenschlager, M., and Fisher, A. G. (2002). Nuclear organisation and gene expression. Curr. Opin. Cell Biol. 14, 372–376. Beerli, R. R., Dreier, B., and Barbas, C. F., III. (2000). Positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl. Acad. Sci. USA 97, 1495–1500. Beerli, R. R., Segal, D. J., Dreier, B., and Barbas, C. F., III. (1998). Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl. Acad. Sci. USA 95, 14628–14633. Belinsky, S. A., Klinge, D. M., Stidley, C. A., Issa, J. P., Herman, J. G., March, T. H., and Baylin, S. B. (2003). Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res. 63, 7089–7093. Belmont, A. S., and Bruce, K. (1994). Visualization of G1 chromosomes: A folded, twisted, supercoiled chromonema model of interphase chromatid structure. J. Cell Biol. 127, 287–302. Bolzer, A., Kreth, G., Solovei, I., Koehler, D., Saracoglu, K., Fauth, C., Muller, S., Eils, R., Cremer, C., Speicher, M. R., et al. (2005). Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes. PLoS Biol. 3, 826–842. Boyle, S., Gilchrist, S., Bridger, J. M., Mahy, N. L., Ellis, J. A., and Bickmore, W. A. (2001). The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells. Hum. Mol. Genet. 10, 211–219. Brink, M. C., van der Velden, Y., de Leeuw, Y., Mateos-Langerak, J., Belmont, A. S., van Driel, R., and Verschure, P. J. (2006). Truncated HP-1 lacking a functional chromatin induces heterochromatinization upon in vivo targeting. Histochem. Cell Biol. 125, 53–61. Brown, K. E., Amoils, S., Horn, J. M., Buckle, V. J., Higgs, D. R., Merkenschlager, M., and Fisher, A. G. (2001). Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells. Nat. Cell Biol. 3, 602–606. Brown, K. E., Guest, S. S., Smale, S. T., Hahm, K., Merkenschlager, M., and Fisher, A. G. (1997). Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin. Cell 91, 845–854. Burke, B., and Stewart, C. L. (2002). Life at the edge: The nuclear envelope and human disease. Nat. Rev. Mol. Cell. Biol. 3, 575–585. Byrd, K., and Corces, V. G. (2003). Visualization of chromatin domains created by the gypsy insulator of Drosophila. J. Cell Biol. 162, 565–574. Cameron, E. E., Bachman, K. E., Myohanen, S., Herman, J. G., and Baylin, S. B. (1999). Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Genet. 21, 103–107. Carmo-Fonseca, M. (2002). The contribution of nuclear compartmentalization to gene regulation. Cell 108, 513–521. Caron, H., van Schaik, B., van der Mee, M., Baas, F., Riggins, G., van Sluis, P., Hermus, M. C., van Asperen, R., Boon, K., Voute, P. A., Heisterkamp, S., van Kampen, A., et al. (2001). The human transcriptome map: Clustering of highly expressed genes in chromosomal domains. Science 291, 1289–1292. Carpenter, A. E., Memedula, S., Plutz, M. J., and Belmont, A. S. (2005). Common effects of acidic activators on large-scale chromatin structure and transcription. Mol. Cell. Biol. 25, 958–968. Carvin, C. D., Parr, R. D., and Kladde, M. P. (2003). Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins. Nucleic Acids Res. 31, 6493–6501.

198

Verschure et al.

Cavalli, G., and Paro, R. (1999). Epigenetic inheritance of active chromatin after removal of the main transactivator. Science 286, 955–958. Cha, J. H. (2000). Transcriptional dysregulation in Huntington’s disease. Trends Neurosci. 23, 387–392. Chambeyron, S., and Bickmore, W. A. (2004). Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription. Genes Dev. 18, 1119–1130. Chastain, P. D., and Sinden, R. R. (1998). CTG repeats associated with human genetic disease are inherently flexible. J. Mol. Biol. 275, 405–411. Ching, R. W., Dellaire, G., Eskiw, C. H., and Bazett-Jones, D. P. (2005). PML bodies: A meeting place for genomic loci? J. Cell Sci. 118, 847–854. Clark, S. J., Harrison, J., and Molloy, P. L. (1997). Sp1 binding is inhibited by (m)Cp(m)CpG methylation. Gene 195, 67–71. Cmarko, D., Verschure, P. J., Martin, T. E., Dahmus, M. E., Krause, S., Fu, X. D., van Driel, R., and Fakan, S. (1999). Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection. Mol. Biol. Cell 10, 211–223. Cmarko, D., Verschure, P. J., Otte, A. R., van Driel, R., and Fakan, S. (2003). Polycomb group gene silencing proteins are concentrated in the perichromatin compartment of the mammalian nucleus. J. Cell Sci. 116, 335–343. Collingwood, T. N., Urnov, F. D., and Wolffe, A. P. (1999). Nuclear receptors: Coactivators, corepressors and chromatin remodeling in the control of transcription. J. Mol. Endocrinol. 23, 255–275. Cosgrove, M. S., Boeke, J. D., and Wolberger, C. (2004). Regulated nucleosome mobility and the histone code. Nat. Struct. Mol. Biol. 11, 1037–1043. Craig, J. M. (2005). Heterochromatin-many flavours, common themes. Bioessays 27, 17–28. Cremer, M., Kupper, K., Wagler, B., Wizelman, L., von Hase, J., Weiland, Y., Kreja, L., Diebold, J., Speicher, M. R., and Cremer, T. (2003). Inheritance of gene density-related higher order chromatin arrangements in normal and tumor cell nuclei. J. Cell Biol. 162, 809–820. Cremer, T., and Cremer, C. (2001). Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat. Rev. Genet. 2, 292–301. Cremer, T., Kupper, K., Dietzel, S., and Fakan, S. (2004). Higher order chromatin architecture in the cell nucleus: On the way from structure to function. Biol. Cell 96, 555–567. Croft, J. A., Bridger, J. M., Boyle, S., Perry, P., Teague, P., and Bickmore, W. A. (1999). Differences in the localization and morphology of chromosomes in the human nucleus. J. Cell Biol. 145, 1119–1131. Daniel, J. A., Pray-Grant, M. G., and Grant, P. A. (2005). Effector proteins for methylated histones: An expanding family. Cell Cycle 4, 919–926. Dehghani, H., Dellaire, G., and Bazett-Jones, D. P. (2005). Organization of chromatin in the interphase mammalian cell. Micron 36, 95–108. Di Croce, L., Raker, V. A., Corsaro, M., Fazi, F., Fanelli, M., Faretta, M., Fuks, F., Lo Coco, F., Kouzarides, T., Nervi, C., Minucci, S., and Pelicci, P. G. (2002). Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 295, 1079–1082. Dillon, N. (2004). Heterochromatin structure and function. Biol. Cell 96, 631–637. Dorigo, B., Schalch, T., Kulangara, A., Duda, S., Schroeder, R. R., and Richmond, T. J. (2004). Nucleosome arrays reveal the two-start organization of the chromatin fiber. Science 306, 1571–1573. Eckey, M., Moehren, U., and Baniamad, A. (2003). Gene silencing by the thyroid hormone receptor. Mol. Cell. Endocrinol. 213, 13–22. Egger, G., Liang, G. N., Aparicio, A., and Jones, P. A. (2004). Epigenetics in human disease and prospects for epigenetic therapy. Nature 429, 457–463.

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

199

Esteller, M. (2005). Aberrant DNA methylation as a cancer-inducing mechanism. Annu. Rev. Pharmacol. Toxicol. 45, 629–656. Esteller, M., Corn, P. G., Baylin, S. B., and Herman, J. G. (2001). A gene hypermethylation profile of human cancer. Cancer Res. 61, 3225–3229. Feinberg, A. P., and Vogelstein, B. (1983). Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301, 89–92. Felsenfeld, G., and Groudine, M. (2003). Controlling the double helix. Nature 421, 448–453. Fischle, W., Wang, Y. M., and Allis, C. D. (2003a). Binary switches and modification cassettes in histone biology and beyond. Nature 425, 475–479. Fischle, W., Wang, Y. M., Jacobs, S. A., Kim, Y. C., Allis, C. D., and Khorasanizadeh, S. (2003b). Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 bv Polvcomb and HP1 chromodomains. Genes Dev. 17, 1870–1881. Fraga, M. F., Ballestar, E., Villar-Garea, A., Boix-Chornet, M., Espada, J., Schotta, G., Bonaldi, T., Haydon, C., Ropero, S., Petrie, K., Iyer, N. G., Perez-Rosado, A., et al. (2005). Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat. Genet. 37, 391–400. Fuks, F., Burgers, W. A., Brehm, A., Hughes-Davies, L., and Kouzarides, T. (2000). DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat. Genet. 24, 88–91. Gall, J. G. (2001). A role for Cajal bodies in assembly of the nuclear transcription machinery. FEBS Lett. 498, 164–167. Gilbert, N., and Allan, J. (2001). Distinctive higher-order chromatin structure at mammalian centromeres. Proc. Natl. Acad. Sci. USA 98, 11949–11954. Goelz, S. E., Vogelstein, B., Hamilton, S. R., and Feinberg, A. P. (1985). Hypomethylation of DNA from benign and malignant human colon neoplasms. Science 228, 187–190. Goldstone, A. P. (2004). Prader-Willi syndrome: Advances in genetics, pathophysiology and treatment. Trends Endocrinol. Metab. 15, 12–20. Grande, M. A., van der Kraan, I., van Steensel, B., Schul, W., de The, H., van der Voort, H. T. M., de Jong, L., and van Driel, R. (1996). PML-containing nuclear bodies: Their spatial distribution in relation to other nuclear components. J. Cell. Biochem. 63, 280–291. Grignani, F., De Matteis, S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce, M., Fanelli, M., Ruthardt, M., Ferrara, F. F., Zamir, I., Seiser, C., Grignani, F., et al. (1998). Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia. Nature 391, 815–818. Gruenbaum, Y., Margalit, A., Goldman, R. D., Shumaker, D. K., and Wilson, K. L. (2005). The nuclear lamina comes of age. Nat. Rev. Mol. Cell. Biol. 6, 21–31. Hall, I. M., Shankaranarayana, G. D., Noma, K. I., Ayoub, N., Cohen, A., and Grewal, S. I. S. (2002). Establishment and maintenance of a heterochromatin domain. Science 297, 2232–2237. Harikrishnan, K. N., Chow, M. Z., Baker, E. K., Pal, S., Bassal, S., Brasacchio, D., Wang, L., Craig, J. M., Jones, P. L., Sif, S., and El-osta, A. (2005). Brahma links the SWI/SNF chromatinremodeling complex with MeCP2-dependent transcriptional silencing. Nat. Genet. 37, 254–264. Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F., and Kemler, R. (2000). The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. EMBO J. 19, 1839–1850. Henikoff, S., Furuyama, T., and Ahmad, K. (2004). Histone variants, nucleosome assembly and epigenetic inheritance. Trends Genet. 20, 320–326. Herman, J. G., and Baylin, S. B. (2003). Gene silencing in cancer in association with promoter hypermethylation. N. Engl. J. Med. 349, 2042–2054. Herrera, F. J., and Triezenberg, S. J. (2004). VP16-dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-early gene promoters during herpes simplex virus infection. J. Virol. 78, 9689–9696.

200

Verschure et al.

Hiratsu, K., Matsui, K., Koyama, T., and Ohme-Takagi, M. (2003). Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34, 733–739. Horn, P. J., and Peterson, C. L. (2002). Molecular biology: Chromatin higher order folding: Wrapping up transcription. Science 297, 1824–1827. Jaenisch, R., and Bird, A. (2003). Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nat. Genet. 33, 245–254. Jaunin, F., Visser, A. E., Cmarko, D., Aten, J. A., and Fakan, S. (2000). Fine structural in situ analysis of nascent DNA movement following DNA replication. Exp. Cell Res. 260, 313–323. Jenuwein, T., and Allis, C. D. (2001). Translating the histone code. Science 293, 1074–1080. Jouvenot, Y., Ginjala, V., Zhang, L., Liu, P. Q., Oshimura, M., Feinberg, A. P., Wolffe, A. P., Ohlsson, R., and Gregory, P. D. (2003). Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors. Gene Ther. 10, 513–522. Kim, V. N. (2005). MicroRNA biogenesis: Coordinated cropping and dicing. Nat. Rev. Mol. Cell. Biol. 6, 376–385. Klose, R., and Bird, A. (2003). Molecular biology. MeCP2 repression goes nonglobal. Science 302, 793–795. Koken, M. H. M., Puvion-Dutilleul, F., Guillemin, M. C., Viron, A., Linares-Cruz, G., Stuurman, N., de Jong, L., Szostecki, C., Calvo, F., and Chomienne, C. (1994). The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion. EMBO J. 13, 1073–1083. Kondo, Y., Shen, L. L., and Issa, J. P. J. (2003). Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer. Mol. Cell. Biol. 23, 206–215. Labrador, M., and Corces, V. G. (2002). Setting the boundaries of chromatin domains and nuclear organization. Cell 111, 151–154. Lachner, M., and Jenuwein, T. (2002). The many faces of histone lysine methylation. Curr. Opin. Cell Biol. 14, 286–298. Lewis, J. D., and Tollervey, D. (2000). Like attracts like: Getting RNA processing together in the nucleus. Science 288, 1385–1389. Li, G., Levitus, M., Bustamante, C., and Widom, J. (2005). Rapid spontaneous accessibility of nucleosomal DNA. Nat. Struct. Mol. Biol. 12, 46–53. Li, G., and Widom, J. (2004). Nucleosomes facilitate their own invasion. Nat. Struct. Mol. Biol. 11, 763–769. Lin, X., Antalffy, B., Kang, D., Orr, H. T., and Zoghbi, H. Y. (2000). Polyglutamine expansion down-regulates specific neuronal genes before pathologic changes in SCA1. Nat. Neurosci. 3, 157–163. Liu, P. Q., Rebar, E. J., Zhang, L., Liu, Q., Jamieson, A. C., Liang, Y., Qi, H., Li, P. X., Chen, B., Mendel, M. C., Zhong, X., Lee, Y. L., et al. (2001). Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J. Biol. Chem. 276, 11323–11334. Luciani, J. J., Depetris, D., Missirian, C., Mignon-Ravix, C., Metzler-Guillemain, C., Megarbane, A., Moncla, A., and Mattei, M. G. (2005). Subcellular distribution of HP1 proteins is altered in ICF syndrome. Eur. J. Hum. Genet. 13, 41–51. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997). Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251–260. Lund, A. H., and van Lohuizen, M. (2004). Epigenetics and cancer. Genes Dev. 18, 2315–2335. Magnenat, L., Blancafort, P., and Barbas, C. F., III. (2004). In vivo selection of combinatorial libraries and designed affinity maturation of polydactyl zinc finger transcription factors for ICAM-1 provides new insights into gene regulation. J. Mol. Biol. 341, 635–649. Maher, E. R., and Reik, W. (2000). Beckwith-Wiedemann syndrome: Imprinting in clusters revisited. J. Clin. Invest. 105, 247–252.

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

201

Mahy, N. L., Perry, P. E., and Bickmore, W. A. (2002a). Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH. J. Cell Biol. 159, 753–763. Mahy, N. L., Perry, P. E., Gilchrist, S., Baldock, R. A., and Bickmore, W. A. (2002b). Spatial organization of active and inactive genes and noncoding DNA within chromosome territories. J. Cell Biol. 157, 579–589. Margolin, J. F., Friedman, J. R., Meyer, W. K., Vissing, H., Thiesen, H. J., and Rauscher, F. J., III. (1994). Kruppel-associated boxes are potent transcriptional repression domains. Proc. Natl. Sci. USA 91, 4509–4513. Margueron, R., Trojer, P., and Reinberg, D. (2005). The key to development: Interpreting the histone code? Curr. Opin. Genet. Dev. 15, 163–176. Martienssen, R. A. (2003). Maintenance of heterochromatin by RNA interference of tandem repeats. Nat. Genet. 35, 213–214. Martinez-Campa, C., Politis, P., Moreau, J. L., Kent, N., Goodall, J., Mellor, J., and Goding, C. R. (2004). Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1. Mol. Cell 15, 69–81. Matsuda, E., Agata, Y., Sugai, M., Katakai, T., Gonda, H., and Shimizu, A. (2001). Targeting of Kruppel-associated box-containing zinc finger proteins to centromeric heterochromatin. Implication for the gene silencing mechanisms. J. Biol. Chem. 276, 14222–14229. Mattout-Drubezki, A., and Gruenbaum, Y. (2003). Dynamic interactions of nuclear lamina proteins with chromatin and transcriptional machinery. Cell. Mol. Life Sci. 60, 2053–2063. Melki, J. (1997). Spinal muscular atrophy. Curr. Opin. Neurol. 10, 381–385. Memedula, S., and Belmont, A. S. (2003). Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16. Curr. Biol. 13, 241–246. Muller, W. G., Walker, D., Hager, G. L., and McNally, J. G. (2001). Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter. J. Cell Biol. 154, 33–48. Mutskov, V. J., Farrell, C. M., Wade, P. A., Wolffe, A. P., and Felsenfeld, G. (2002). The barrier function of an insulator couples high histone acetylation levels with specific protection of promoter DNA from methylation. Genes Dev. 16, 1540–1554. Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D., and Grewal, S. I. (2001). Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113. Nicholls, R. D., Saitoh, S., and Horsthemke, B. (1998). Imprinting in Prader-Willi and Angelman syndromes. Trends Genet. 14, 194–200. Nicole, S., Diaz, C. C., Frugier, T., and Melki, J. (2002). Spinal muscular atrophy: Recent advances and future prospects. Muscle Nerve 26, 4–13. Noma, K., Allis, C. D., and Grewal, S. I. (2001). Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries. Science 293, 1150–1155. Nye, A. C., Rajendran, R. R., Stenoien, D. L., Mancini, M. A., Katzenellenbogen, B. S., and Belmont, A. S. (2002). Alteration of large-scale chromatin structure by estrogen receptor. Mol. Cell. Biol. 22, 3437–3449. Ogg, S. C., and Lamond, A. I. (2002). Cajal bodies and coiling: Moving towards function. J. Cell Biol. 159, 17–21. Oostra, B. A., and Willemsen, R. (2002). The X chromosome and fragile X mental retardation. Cytogenet. Genome Res. 99, 257–264. Orr, H. T. (2001). Beyond the Qs in the polyglutamine diseases. Genes Dev. 15, 925–932. Pandolfi, P. P. (2001). Histone deacetylases and transcriptional therapy with their inhibitors. Cancer Chemother. Pharmacol. 48, S17–S19. Parada, L. A., and Misteli, T. (2002). Chromosome positioning in the interphase, nucleus. Trends Cell Biol. 12, 425–432.

202

Verschure et al.

Pengue, G., and Lania, L. (1996). Kruppel-associated box-mediated repression of RNA polymerase II promotors is influenced by the arrangement of basal promotor elements. Proc. Natl. Acad. Sci. USA 93, 1015–1020. Perez-Burgos, L., Peters, A. H., Opravil, S., Kauer, M., Mechtler, K., and Jenuwein, T. (2004). Generation and characterization of methyl-lysine histone antibodies. Methods Enzymol. 376, 234–254. Peters, A. H. F. M., Kubicek, S., Mechtler, K., O’Sullivan, R. J., Derijck, A. A. H. A., Perez-Burgos, L., Kohimaier, A., Opravil, S., Tachibana, M., Shinkai, Y., Martens, J. H., Jenuwein, T., et al. (2003). Partitioning and plasticity of repressive histone methylation states in mammalian chromatin. Mol. Cell 12, 1577–1589. Rea, S., Eisenhaber, F., O’Carroll, D., Strahl, B. D., Sun, Z. W., Schmid, M., Opravil, S., Mechtler, K., Ponting, C. P., Allis, C. D., and Jenuwein, T. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593–599. Reinhart, B. J., and Bartel, D. P. (2002). Small RNAs correspond to centromere heterochromatic repeats. Science 297, 1831. Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., Volkert, T. L., Wilson, C. J., et al. (2000). Genome-wide location and function of DNA binding proteins. Science 290, 2306–2309. Rice, J. C., and Allis, C. D. (2001). Histone methylation versus histone acetylation: New insights into epigenetic regulation. Curr. Opin. Cell Biol. 13, 263–273. Rice, J. C., Briggs, S. D., Ueberheide, B., Barber, C. M., Shabanowitz, J., Hunt, D. F., Shinkai, Y., and Allis, C. D. (2003). Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains. Mol. Cell 12, 1591–1598. Robinett, C. C., Straight, A., Li, G., Willhelm, C., Sudlow, G., Murray, A., and Belmont, A. S. (1996). In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J. Cell Biol. 135, 1685–1700. Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988). GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 564. Salozhin, S. V., Prokhorchuk, E. B., and Georgiev, G. P. (2005). Methylation of DNA—one of the major epigenetic markers. Biochemistry (Mosc.) 70, 525–532. Schmitz, M. L., Stelzer, G., Altmann, H., Meisterernst, M., and Baeuerle, P. A. (1995). Interaction of the COOH-terminal transactivation domain of p65 NF-kappa B with TATA-binding protein, transcription factor IIB, and coactivators. J. Biol. Chem. 270, 7219–7226. Schultz, D. C., Ayyanathan, K., Negorev, D., Maul, G. G., and Rauscher, F. J., III. (2002). SETDB1: A novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev. 16, 919–932. Shiels, C., Islam, S. A., Vatcheva, R., Sasieni, P., Sternberg, M. J., Freemont, P. S., and Sheer, D. (2001). PML bodies associate specifically with the MHC gene cluster in interphase nuclei. J. Cell Sci. 114, 3705–3716. Shopland, L. S., Johnson, C. V., Byron, M., McNeil, J., and Lawrence, J. B. (2003). Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: Evidence for local euchromatic neighborhoods. J. Cell Biol. 162, 981–990. Snowden, A. W., Gregory, P. D., Case, C. C., and Pabo, C. O. (2002). Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr. Biol. 12, 2159–2166. Snowden, A. W., Zhang, L., Urnov, F., Dent, C., Jouvenot, Y., Zhong, X., Rebar, E. J., Jamieson, A. C., Zhang, H. S., Tan, S., Case, C. C., Pabo, C. O., et al. (2003). Repression of vascular endothelial growth factor A in glioblastoma cells using engineered zinc finger transcription factors. Cancer Res. 63, 8968–8976.

5. Epigenetic Gene Control Systems and Engineered Transcription Factors

203

Spector, D. L. (2001). Nuclear domains. J. Cell Sci. 114, 2891–2893. Stege, J. T., Guan, X., Ho, T., Beachy, R. N., and Barbas, C. F., III. (2002). Controlling gene expression in plants using synthetic zinc finger transcription factors. Plant J. 32, 1077–1086. Sun, F. L., Cuaycong, M. H., and Elgin, S. C. (2001). Long-range nucleosome ordering is associated with gene silencing in Drosophila melanogaster pericentric heterochromatin. Mol. Cell. Biol. 21, 2867–2879. Sutcliffe, J. S., Nelson, D. L., Zhang, F., Pieretti, M., Caskey, C. T., Saxe, D., and Warren, S. T. (1992). DNA methylation represses FMR-1 transcription in fragile X syndrome. Hum. Mol. Genet. 1, 397–400. Tachikawa, K., Schroder, O., Frey, G., Briggs, S. P., and Sera, T. (2004). Regulation of the endogenous VEGF-A gene by exogenous designed regulatory proteins. Proc. Natl. Acad. Sci. USA 101, 15225–15230. Taddei, A., Roche, D., Bickmore, W. A., and Almouzni, G. (2005). The effects of histone deacetylase inhibitors on heterochromatin: Implications for anticancer therapy? EMBO Rep. 6, 520–524. Tan, S., Guschin, D., Davalos, A., Lee, Y. L., Snowden, A. W., Jouvenot, Y., Zhang, H. S., Howes, K., McNamara, A. R., Lai, A., Ullman, C., Reynolds, L., et al. (2003). Zinc-finger proteintargeted gene regulation: Genomewide single-gene specificity. Proc. Natl. Acad. Sci. USA 100, 11997–12002. Tanabe, H., Habermann, F. A., Solovei, I., Cremer, M., and Cremer, T. (2002). Non-random radial arrangements of interphase chromosome territories: Evolutionary considerations and functional implications. Mutat. Res. 504, 37–45. Thiel, G., Lietz, M., and Hohl, M. (2004). How mammalian transcriptional repressors work. Eur. J. Biochem. 271, 2855–2862. Tufarelli, C., Stanley, J. A., Garrick, D., Sharpe, J. A., Ayyub, H., Wood, W. G., and Higgs, D. R. (2003). Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat. Genet. 34, 157–165. Tumbar, T., and Belmont, A. S. (2001). Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator. Nat. Cell Biol. 3, 134–139. Tumbar, T., Sudlow, G., and Belmont, A. S. (1999). Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J. Cell Biol. 145, 1341–1354. Turner, B. M. (2002). Cellular memory and the histone code. Cell 111, 285–291. Urnov, F. D. (2002). A feel for the template: Zinc finger protein transcription factors and chromatin. Biochem. Cell Biol. 80, 321–333. Urnov, F. D. (2003). Chromatin remodeling as a guide to transcriptional regulatory networks in mammals. J. Cell Biochem. 88, 684–694. Urnov, F. D., Yee, J., Sachs, L., Collingwood, T. N., Bauer, A., Beug, H., Shi, Y. B., and Wolffe, A. P. (2000). Targeting of N-CoR and histone deacetylase 3 by the oncoprotein v-erbA yields a chromatin infrastructure-dependent transcriptional repression pathway. EMBO J. 19, 4074–4090. Urrutia, R. (2003). KRAB-containing zinc-finger repressor proteins. Genome Biol. 4, 231. Verdel, A., Jia, S. T., Gerber, S., Sugiyama, T., Gygi, S., Grewal, S. I. S., and Moazed, D. (2004). RNAi-mediated targeting of heterochromatin by the RITS complex. Science 303, 672–676. Verschure, P. J. (2004). Positioning the genome within the nucleus. Biol. Cell 96, 569–577. Verschure, P. J., van der Kraan, I., de Leeuw, W., van der Vlag, J., Carpenter, A. E., Belmont, A. S., and van Driel, R. (2005). In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Mol. Cell. Biol. 25, 4552–4564. Verschure, P. J., van der Kraan, I., Manders, E. M., Hoogstraten, D., Houtsmuller, A. B., and van Driel, R. (2003). Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules. EMBO Rep. 4, 861–866.

204

Verschure et al.

Verschure, P. J., van der Kraan, I., Enserink, J. M., Mone, M. J., Manders, E. M., and van Driel, R. (2002). Large-scale chromatin organization and the localization of proteins involved in gene expression in human cells. J. Histochem. Cytochem. 50, 1303–1312. Verschure, P. J., van der Kraan, I., Manders, E. M., and van Driel, R. (1999). Spatial relationship between transcription sites and chromosome territories. J. Cell Biol. 147, 13–24. Versteeg, R., van Schaik, B. D., van Batenburg, M. F., Roos, M., Monajemi, R., Caron, H., Bussemaker, H. J., and van Kampen, A. H. (2003). The human transcriptome map reveals extremes in gene density, intron length, GC content, and repeat pattern for domains of highly and weakly expressed genes. Genome Res. 13, 1998–2004. Visser, A. E., and Aten, J. A. (1999). Chromosomes as well as chromosomal subdomains constitute distinct units in interphase nuclei. J. Cell Sci. 112, 3353–3360. Visser, A. E., Jaunin, F., Fakan, S., and Aten, J. A. (2000). High resolution analysis of interphase chromosome domains. J. Cell Sci. 113, 2585–2593. Volpe, T. A., Kidner, C., Hall, I. M., Teng, G., Grewal, S. I. S., and Martienssen, R. A. (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837. Volpi, E. V., Chevret, E., Jones, T., Vatcheva, R., Williamson, J., Beck, S., Campbell, R. D., Goldsworthy, M., Powis, S. H., Ragoussis, J., Trowsdale, J., and Sheer, D. (2000). Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei. J. Cell Sci. 113, 1565–1576. Wallrath, L. L., and Elgin, S. C. R. (1995). Position effect variegation in Drosophila is associated with an altered chromatine structure. Genes Dev. 9, 1263–1277. Wang, J., Shiels, C., Sasieni, P., Wu, P. J., Islam, S. A., Freemont, P. S., and Sheer, D. (2004a). Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions. J. Cell Biol. 164, 515–526. Wang, Y., Fischle, W., Cheung, W., Jacobs, S., Khorasanizadeh, S., and Allis, C. D. (2004b). Beyond the double helix: Writing and reading the histone code. Novartis Found. Symp. 259, 3–17. Weintraub, H., and Groudine, M. (1976). Chromosomal subunits in active genes have an altered conformation. Science 193, 848–856. Williams, R. R., Broad, S., Sheer, D., and Ragoussis, J. (2002). Subchromosomal positioning of the epidermal differentiation complex (EDC) in keratinocyte and lymphoblast interphase nuclei. Exp. Cell Res. 272, 163–175. Yamamoto, A., Lucas, J. J., and Hen, R. (2000). Reversal of neuropathology and motor dysfunction in a conditional model of Huntington’s disease. Cell 101, 57–66. Yuan, G. C., Liu, Y. J., Dion, M. F., Slack, M. D., Wu, L. F., Altschuler, S. J., and Rando, O. J. (2005). Genome-scale identification of nucleosome positions in S. cerevisiae. Science 309, 626–630. Zhang, L., Spratt, S. K., Liu, Q., Johnstone, B., Qi, H., Raschke, E. E., Jamieson, A. C., Rebar, E. J., Wolffe, A. P., and Case, C. C. (2000). Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J. Biol. Chem. 275, 33850–33860. Zhang, Y., and Reinberg, D. (2001). Transcription regulation by histone methylation: Interplay between different covalent modifications of the core histone tails. Genes Dev. 15, 2343–2360. Zhong, S., Hu, P., Ye, T. Z., Stan, R., Ellis, N. A., and Pandolfi, P. P. (1999). A role for PML and the nuclear body in genomic stability. Oncogene 18, 7941–7947. Zimber, A., Nguyen, Q. D., and Gespach, C. (2004). Nuclear bodies and compartments: Functional roles and cellular signalling in health and disease. Cell. Signal. 16, 1085–1104. Zoghbi, H. Y., and Orr, H. T. (2000). Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci. 23, 217–247.

Index A Abnormalities, epigenetic, 132 Acetylation, 133 Achaete-scute complex (ASC), 65 Activation, transcriptional, 138 Allele(s) female transmitted Igf2, 81 male harboring, 65 Allelomorph, 58 Allelomorphism, step, 64 Amide linkage, 12 Aminoacetaldehyde, reductive amination of N-protected, 5 Amino acids chiral, 9 Boc-protected, 5 Angelman syndrome, 80, 179 Angiogenesis therapeutic, 146 tumor, 145 Ascialo glycoprotein receptor (ASGP-R), 17 Ataxia, spinocerebellar, 181 B Bacteria, pathogenic, 18 Beckwith-Wiedemann syndrome, 80, 179 Benzyloxycarbonyl (Cbz) protection, 5 Binding high affinity –of TFOs, 37 –of ZF-TF to naked DNA, 155 in vivo natural DNA, factors, 193 ligand, to specific cell-surface receptors, 31 major-groove, ZFPs, 151 prevention of, of essential transcription factors, 134 transcription-factor, motifs, 151 ZFP, and nucleosomal structure, 148 ZF-TF, 148

Biostability, 2 Biosynthesis, chlorophyll, 76 BisPNA, 8 Bonding, Hoogsten, 8 Bovine spongiform encephalopathy (BSE), 84 Bromoacetate, alkyl, 5 BSE. See Bovine spongiform encephalopathy Burkitt’s lymphoma cell lines, 27 C Cancer, biology, 179 epigenetics, 179 Cardiomyopathy, 77 Cell-penetrating peptides (CPP), 7 Cell walls, composition and structure of bacterial, 18 Checkpoint kinase 2 (CHK2), 141 Chimera, periclinal, 115, 117 –characteristics of, 122 production of, graft hybrids, 114 Chlorophyll biosynthesis, 76 Chromatin, active, 176f changes in, structure, 170 compact, 174 condensed, 174 decondensation, 171–172 –of, structure, 184 digestion of, by DNase I, 152 domains, 170 functional, domains, 169–170 immunoprecipitation (ChIP), 152 in vivo visualization of, fibers, 171 looping out of a compact, domain, 174 Chromonema fibers, 169 Chromosomal rearrangements, 133–134 Chromosome, 175 breakage, 80 205

Index

206 Chromosome (cont. ) nuclei, 175 pachytene, 74 paternal, 62 salivary, bands, 74 theory, 57 –of heredity, 113 Control, dysregulated long range, 133 Creutzfeldt–Jakob disease, 84–85 Cross-pollination, 115 CXCR3 chemokine receptor, 21 Cytogenetics, 54 Cytoplasmic organelles, 54 D Dexametasone, 32 Direct targeting of HP1, 187 Disease(s), Creutzfeldt–Jakob, 84–85 dysregulated long range control, due to, 133 hereditary, 77 human, and epigenetic gene control mechanisms, 177 human, relevant to mitochondria, 76 Huntington’s, 181 mitochondrial, 77 monogenetic, 136 Parkinson, 77 severe combined immunodeficiency, (SCID), 136 sickle cell, 147 DNA, 2–3, 10, 54, 180 accessibility of, to regulatory proteins, 166 bacterial, immunostimmulatory effect of, 32 base composition of, 61 binding, 10 –affinity, 12, 154 –agents, 155 –domain, 138 –molecules, gene-specific, 137 –molecules, sequence specific, 139 –of PNA-complexes to, 27 –zinc-finger, domains, 144, 182 chloroplast –in plants, 76 –replication, 76 defects at level of, 132

displaced, strand, 24 double stranded, 3, 8 effect of, hypomethylation on hetereochromatin organization, 181 genomic –in eukaryotes, 75 –and plasmid, 3 ligases, 67 methylation, 37, 82, 132, 165, 169, 180 –in germ cells, 179 –patterns on DNA, 134 methyltransferases, 83 molecular interactions with, 167 negatively supercoiled, 26 nucleosomal, 150 polymerase elongation, 27 recombinant, technologies, 86 replication, 172, 174 single-stranded –loop (D-loop), 24 –molecules, 140 site-specific digestion of, 36 specific triplets of, 141 targeting of, for downregulation of gene expression, 155 vaccination, 32 wrapping of, around the nucleosome, 166 DNase hypersensitive sites, 152–153 Domain activation, 141, 185 –effector, 182 effector, 156, 190 gene-activation and -repression, 194 intrapolypeptide, 69 repressor, 141 transcriptional activity of, 171 Downregulation of gene(s), 134 of gene expression, 146 of RNA, 27–28 Doxorubicin, antitumor drug, 144 Drosophila protein Antennapedia, 16 Dystrophy, myotonic, 181 E Electroporation, 25 Encephalitis, scrapie, of sheep, 83 Encephalopathy, 77 Endonucleases, restriction, 67

Index Enzyme chromatin-modifying, 182 chromatin-remodeling, 186 histone-modifying, 190 one gene–one enzyme concept, 56 Epigenesis, 78 Epigenetic information, 164–165 Epigenetic modification of gene expression, 81 Epigenetic therapy, 134 Epimutations, meiotic transmission of, 82 Epistasis, 72 Erythropoietin (Epo), 147 Ethylenediamine, alkylation of, 5 Euchromatin, 170 Eukaryotic translation termination complex (eRF3), 86 F Fertility, low-pollen, 115 Fertilization, mechanism of, 54 FISH-protocols, 35 Fluorenylmethoxycarbonyl (Fmoc) strategy, 5 Fragile-X syndrome, FMR1 gene in, 180 Fruit trees, heredity of grafted, 121 G Gene. See also Genome activation, 191 –of imprinted, 192 adenosine deaminase, 136 advantage of, therapy, 137 chemical nature of, 59 clinical, therapy, 136 control, 194 in cytoplasm, 75 definition of, 62 downregulation of, expression, 146 dysregulated, expression, 132 endogenous –expression, 140 –modulation, 155 –targeted for upregulation, 147 epigenetic –control, 179, 182 –control mechanisms, 165, 167 –control systems, 145 –regulation, 181 expression, 132

207

human prion, 85f genetic dissection of mitochondrial, functions, 76 genome-wide analysis of, expression, 70 higher orders of, 70 immunoglobulin, 68 inducing endogenous, expression, 138 maternal-repressed IGF2, 147 modulation –of, Expression by Engineered Zinc-Finger Transcription factors, 143 –of, expression via epigenetic control mechanisms, 166 molecular units of, 68 nonsexual transfer of, 67 number, 73 physical concepts of, 57 repression, 167, 186 –mechanisms, 190 silencing, 63, 189, 191 –and activation, 82 single nucleic acid, 54 size, 73 structure of, 64 successful, therapy of LHON, 77 therapy, 30, 136 –trials, 136 transcription, 26 –in vivo, 27 upregulation, 191 –of endogenous, expression by engineered ZFPs, 146 Genetic code, 63 Genetic information, 164–165 Genetics, 56 reverse, 135 Genome division of, 171f functional organization of human, 134 higher-order, 164 human, 174 sequence data, 73 sequencing, 74 Gerstmann–Straussler syndrome, 84–85 Glucocorticoid receptor (GR), 32, 172 Glycine N-(2-aminoethyl), 2 replacement of, within aeg-PNA, 12 Glycosylphosphatidyl inositol anchor (GPI), 84

Index

208 Graft. See also Grafting chimera, 105, 111 hybrid, 102–104, 111 hybridization, 102–105, 110, 122–125 in vitro, techniques, 114 propagation, 110, 119 transfer of cytoplasmic male sterility, 108 Grafting, 102 hereditary changes associated with, 109 induction of genetic changes by repeated, 109 mentor, 114 –method, 110, 112, 115 –methodology, 122 Michurin’s mentor, method, 111 H Happy puppet syndrome. See Angelman syndrome HeLa-cell nuclear extract, 25 Hepatitis C Virus (HBV), 23 Hereditary diseases, 77 Herpes simplex viral VP16, 146 Herpes simplex virus transactivating protein VP16, 182 Heterochromatin, 70 alterations in structure of, 180 binding proteins, 193 constitutive, 170 facultative, 170, 186 protein, 172 Heterochromatinization, 191 Heteroplasmy, 76 Histone, methylation, 169 methyltransferases, 193 modification, 133, 168–169 monoacetylation and trimethylation of, H4, 180 HIV revresponse element (RRE), 23 HIV-1 infected lymphoma cell line, 13 Homooligomers, 10 Hoogsten bonding, 8 Hormone metabolisms, 76 Human telomeres (50 -TTAGGG-30), 23 Human tumor cells, 180 Huntington’s chorea, 77. See also Disease, Huntington’s

Hybrid chimera graft, 111, 115, 117 existence, –of graft, 107, 109 –of somatic, 102 formation, –of graft, 104, 116 –of nonchimera graft, 117 –of, by grafting, 107 fusion graft, 105 nonchimera graft, 117 seedling, 123 sexual, 105 systems based on nonchimera graft, 115 Hybridization. See also Hybrid characteristics of graft, 115–116 conception of graft, 103 fluorescence in situ, (FISH), 3 intergeneric, 123 mechanism of graft, 113, 116, 118 methods of graft, 114 Michurin’s graft, 112 –contribution to, 106 microinjection of preformed plasmid-PNA, complexes, 24 nucleic-acid, 32 plasmid, 31 PNA–DNA, 35 sexual, 103, 123 significance of graft, 120 Hypermethylation of CpG islands, 180 I ICF syndrome, 180 Idioplasm, 59 Imunodeficiency, 137 Imprinting, 79, 179 Infections, cytomegalovirus, of eye, 135 Insomnia, fatal familial, 85 Insulin-like growth factor 1 receptor (IGF1-R), 17 Insulin-like growth factor II (IGF2), 80 Itercellular adhesion molecule (ICAM-1), 147 Internal ribosomal entry sites (IRES), 22 Intravenous administration of anti-CXCR3PNA, 21 Introns, 70

Index K KRAB. See Kruppel-associated box Kruppel-associated box domains, 186 repression domain, 144, 153, 187 L Lamarck’s hypothesis, 105 Leber’s hereditary optic atropy (LHON), 77 Leukemia, 137 Light-up probes, 33 Lipofectamine, 18 Lipophilic conjugates, uptake via liposomes and, 17 Lipophilic triphenylphosphonium cation (TPP), 18 Lipoplexmediated transfection of nondividing cells, 32 Liposomes, cationic, 25 Locked nucleic acid (LNA), 31 M Meiosis, 78 Mendel’s laws of heredity, 121, 124 Methionine codons, 64 Methylation, 133 of cytosine, 167 – in CpG dinucleotides, 167 and enzyme digestion, 36 lysine, 168 of target genes, 81 Micrococcal nuclease, 153 Molecular beacons, 32–33 Monkey kidney fibroblast cells, 24 mRNA effects on, splice site selection, 22 human, 22 mtDNA, analysis of, samples, 77 Mutation affecting chloroplast genes, 76 discovery of induction of, 55 Drosophila genes by X-ray induction, 57 in histone acetyltransferase, 133 in human chromosome, 80 in transcription factors, 133 spontaneous, 109 Mutational hit theory, 56

209

Myoclonic epilepsy associated with ragged-red fibers (MERRF), 77 Myopathy, mitochondrial, 77 N Neovascularization, 146–147 Neurodegenerative disorders, 181 NLS. See Nuclear localization signal Nuclear localization signal, 17 peptides, 30 containing bioplex, 31 Nucleic acid, 60 peptide conjugates, 12 Nucleosomal packaging, 166 Nucleosome, 148 crystal structure of, 149f Nucleotides, eukaryotic coding, 75 O Oligodeoxyribonucleic acids, antisense, (ODNs), 135 Oligomers 20 -O-methoxyethyl (20 -O-MOE) antisense, 22 20 -O-methyl RNA, 23 phosphorothioate, 23 PNA, 21 synthesis of, 5 Oligonucleotides, 12, 32 amide linked, 12 Oligos, triplex forming, 140 Ophthalmoplegia, 77 Organelles, cytoplasmic, 54 P Pangenesis, 102 Darwin’s, 120, 124–125 Parkinson disease, 77 Paramutation, 78 PCR amplification of ligated material, 36 clamping, 35 Pentapeptide, ribosomal subunit, 74 Peptide commercial, synthesizers, 4 cationic. See Cell-penetrating peptides chemical linkage of NLS, 30 cystein-containing, 31

210 Peptide (cont. ) synthesis, 4 uptake of PNA conjugated to cell-penetrating, 16 P-glycoprotein expression, 144 Phosphonate linkage, 12 Plant breeding, 122 chimeras, 120–121 Plasmid cell-specific uptake of, 31 transfection of hybridized, 25 Pleiotropic defects, 77 Peptide nucleic acid, 2–4, 7, 10, 12, 18, 33, 36 affinity between, and natural nucleic acids, 29 alpha-helical, 12 aminoethylglycine, (aeg-PNA), 2, 9 aminoethylpropyl, (aep-PNA), 10 anchors acting as genetic glue, 29 antigene effects of, 26 antitelomerase, 23 artificial restriction enzymes, 36 assembly process of, 4 assisted rare cleavage (PARC), 37 based artificial ribonucleases, 19 beacons, 32, 35 binding kinetics of linear, peptides, 31 biological effects of, administration in vivo, 13 biotechnological aspects of, 29 cationic, 9, 12 cellular, –delivery of, to eukaryotic cells ex vivo, 14t –uptake of unmodified, in vitro, 13 chemical modifications in, 3 chemistry and structure, 4 construction of tail-clamp, 26 containing ‘‘decoy’’ transcription factor responsive elements, 28 custom made, 5 cyclohexyl, 10 different fluorescent, probes, 34f effects –of, and PNA-conjugates in vivo, 38 –on RNA, 19 feature of unmodified, 13 fluorinated olefin, (F-OPA), 12 future prospects for, as antisense and antigene drugs, 28

Index future use of, in vitro, 38 in gene regulation, 24 hybridization to DNA and RNA, 32, 35 induced targeted mutagenesis, 37 inhibition of bacterial mRNAs, 21 localization of, 20 at RNA-level, 21 mechanism of action for, on RNA, 19 melting processes of single-stranded, 7 modified, 10 nucleic acid –complexes, 7 –duplexes, 7 –triplexes, 7 nuclear uptake of, 262 passive diffusion of, over lipid membranes, 13 PNA hybridization, 29 processes leading to, complex formation, 8 recognition of cellular RNAs, 19 of RNA, 19 RNA duplexes, 7 and RNA localization, 20 single-stranded, 7–8 solution properties of, 6 strand invasion, 27 synthesis of, 4 tissue distribution and pharmacokinetic parameters for an unmodified, 16 transferrin-conjugated, 31 uptake –of, in eukaryotic cells, 13 –of, targeted to specific cellular receptors, 17 use of, to detect PCR products, 33 PNA. See Peptide nucleic acid Polyamides DNA-binding, 139 minor-groove binding, 151 Polymerization, PrPSc-induced amyloid, reactions, 84 Polymorphisms, single nucleotide, 58 Polypeptides, availability of toxic, 181 Prader-Willi syndrome, 80, 179 Prion forming domain (PrD), 84 protein infectious agents, 83 protease resistant infective, 83 Prokaryotes, 67, 74

Index Proliferating cell nuclear antigen (PCNA), 83 Proliferation, deregulation of, 180 Promyelocytic leukemia bodies (PML), 175 Protein interactions, 72 amyloid precursor, (APP), 21 optimization of DNA-binding specificity by, engineering, 147 cytoplasmic prion, 85 DNA-binding, 36 –zinc-finger, 150 localization of HP1, 181 pleiotropic nature of regulatory, 73 sequence-specific DNA-binding, 137 Pseudoallelism, 56, 64, 66f Pseudo-isocytosines, 9 Pyrrolidine-methyl-thymine-1-acetyl-glycinePNA (pmg-PNA), 10 Q Quantitative trait loci (QTL), 72 R Regions of increased gene expression (RIDGES), 174 Repression domain, 138, 186 –effector, 186 of endogenous VEGF-A expression, 145 of vErbA, 190 transcriptional, 187 Residues, 4-aminoproline-based, 10 Retrotransposons, 119 Retroviruses, 119, 136 Rett syndrome, 134 Ribonucleoprotein inhibition of, complexes, 23 small, gene, 80 Ribosomes, 23 RNA, 2, 10, 54 antisense molecules decrease, 135 binding, 10 cellular, 19 double-stranded, 29 effects on messenger, 20 genes, 62 genomes, 22 –dimerization, 22

211

higher order, structures, 20 induced silencing complexes (RISCs), 135 inhibition of viral, 22 inhibitor, 19 messenger, expression using PNA, 20 noncoding, 28 polymerase II, 133 processing, 19 ribosomal, 23 –transcription, 177 RNP activities, 19 Rosetta Stone hypothesis, 69 S Silencing induction of gene-specific, 189 posttranscriptional gene, 135 repeat-induced RNAi-mediated, 82 of reporter plasmid, 187 siRNA, administration of, 136 SNPRN, 80 Splicing, alternative, 69 Spinocerebellar ataxia, 181 Staining, cyanine dye, of PNA–DNA duplexes, 35 Step allelomorphism, 64 Stereoplasm, 59 Steroid-mediated gene delivery (SMGD), 32 T Tert-butoxycarbonyl (Boc) protection, 5 TFs, Zinc-finger based, 141 Thyroid hormone receptor (TR), 188 Toll-like receptors (TLRs), stimulation of, 29 Transcript, antisense, 80 Transcription associated factors (TAF), 73 binding sites of known, factors, 153 engineered, factors, 139, 146 gene, 139 of green fluorescence protein (GFP), 24, 150 genetic manipulation of engineered, factors, 138 initiation, 69 –by a PNA-anchored artificial activation domain, 25 –from PNA-displaced ssDNA loops, 24

Index

212 Transfection complexes, cell-specific uptake of, 31 Transmissible spongiform encephalopathies (TSE), 83 Transmission of imprinted signals during embryonic development, 179 Triplexes, stabilization of, 10 Tryptophan, 64 Tumor cell, inhibition of, proliferation, 141 Tumorigenesis, H19 gene, 81

downregulation of overexpressed, 192 gene, 152 induced upregulation of, 146 VP64 activation domain, 153

W Watson–Crick base pairing, 4, 8

Z V Vascular endothelial cadherin protein (CDH5), 147 VEGF-A

ZF-TF effects on epigenetic gene regulation, 181 Zinc-finger protein gene, ZNF127, 80 Zinc-finger recognition sites, 155