International Review Of Cell and Molecular Biology, Volume 282 (International Review of Cell & Molecular Biology)

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INTERNATIONAL REVIEW OF

CELL AND MOLECULAR BIOLOGY

INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY Series Editors

GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN FRIEDLANDER JONATHAN JARVIK

1949–1988 1949–1984 1967– 1984–1992 1993–1995

Editorial Advisory Board

ISAIAH ARKIN PETER L. BEECH ROBERT A. BLOODGOOD DEAN BOK KEITH BURRIDGE HIROO FUKUDA RAY H. GAVIN MAY GRIFFITH WILLIAM R. JEFFERY KEITH LATHAM

WALLACE F. MARSHALL BRUCE D. MCKEE MICHAEL MELKONIAN KEITH E. MOSTOV ANDREAS OKSCHE MANFRED SCHLIWA TERUO SHIMMEN ROBERT A. SMITH ALEXEY TOMILIN

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INTERNATIONAL REVIEW OF

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KWANG W. JEON Department of Biochemistry University of Tennessee Knoxville, Tennessee

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

Front Cover Photography: Cover figure by Jacques Rouquette Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 32 Jamestown Road, London NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands First edition 2010 Copyright # 2010, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@elsevier. com. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material. Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress For information on all Academic Press publications visit our website at elsevierdirect.com

ISBN: 978-0-12-381256-8

PRINTED AND BOUND IN USA 10 11 12 10 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors

1. Functional Nuclear Architecture Studied by Microscopy: Present and Future

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Jacques Rouquette, Christoph Cremer, Thomas Cremer, and Stanislav Fakan 1. Introduction 2. Historical Background 3. Models of Nuclear Architecture and Experimental Evidence 4. Perspectives: Open Questions and Possibilities to Answer Them 5. Quantitative Microscopic Analysis of Nuclear Architecture 6. Concluding Remarks Acknowledgments References

2. Meiotic Silencing in Caenorhabditis elegans

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Eleanor M. Maine 1. 2. 3. 4. 5.

Introduction Chromatin Regulation in the Germ Line Repressive Mechanisms in C. elegans Meiotic Germ Line Meiotic Silencing and Germ Line Development in C. elegans Mechanistic and Functional Comparison of Meiotic Silencing Phenomena in Different Species 6. Noncoding RNA and Chromatin Structure 7. Conclusions and Future Prospects Acknowledgments References

3. Lipid Rafts, Caveolae, and Their Endocytosis

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Patrick Lajoie and Ivan R. Nabi 1. Introduction 2. The Lipid Raft 3. Caveolae

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4. Raft-Dependent Endocytosis 5. Conclusion References

4. New Insights into the Mechanism of Fibroblast to Myofibroblast Transformation and Associated Pathologies

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Mitchell A. Watsky, Karl T. Weber, Yao Sun, and Arnold Postlethwaite 1. 2. 3. 4. 5.

Introduction Fibroblasts and Intermediates Myofibroblasts Fibroblast to Myofibroblast Signaling Mononuclear Cells in Peripheral Blood That Differentiate into Myofibroblasts 6. Myofibroblast-Associated Pathologies 7. Summary References

Index

166 167 169 170 172 176 183 184 193

CONTRIBUTORS

Christoph Cremer Biophysics of Genome Structure, Institute for Pharmacy and Molecular Biotechnology and Kirchhoff-Institute for Physics, University Heidelberg, Heidelberg, Germany Thomas Cremer Biocenter, Ludwig Maximilians University (LMU), Martinsried, Germany, and Munich Center for Integrated Protein Science, Munich, Germany Stanislav Fakan Biocenter, Ludwig Maximilians University (LMU), Martinsried, Germany Patrick Lajoie Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, USA Eleanor M. Maine Department of Biology, Syracuse University, Syracuse, New York, USA Ivan R. Nabi Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada Arnold Postlethwaite Division of Rheumatology, Department of Medicine, University of Tennessee Health Science Center, and Veterans Affairs Medical Center, Memphis, Tennessee, USA Jacques Rouquette Biocenter, Ludwig Maximilians University (LMU), Martinsried, Germany Yao Sun Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA Mitchell A. Watsky Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, USA Karl T. Weber Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA vii

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C H A P T E R

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Functional Nuclear Architecture Studied by Microscopy: Present and Future Jacques Rouquette,*,1 Christoph Cremer,† Thomas Cremer,*,‡ and Stanislav Fakan* Contents 1. Introduction 2. Historical Background 3. Models of Nuclear Architecture and Experimental Evidence 3.1. Current models 3.2. Experimental evidence 4. Perspectives: Open Questions and Possibilities to Answer Them 4.1. Studies of higher order chromatin arrangements 4.2. Nuclear topography and models of the nuclear architecture 4.3. Forces responsible for changes of higher order chromatin arrangements 4.4. Unexplored biophysical properties of nuclei 4.5. Summary 5. Quantitative Microscopic Analysis of Nuclear Architecture 5.1. Electron microscopy 5.2. Far-field fluorescence microscopy with conventional resolution 5.3. Far-field fluorescence microscopy with resolution beyond the Abbe limit 5.4. Importance of correlation microscopy for new ways to realize an old concept 6. Concluding Remarks Acknowledgments References

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* Biocenter, Ludwig Maximilians University (LMU), Martinsried, Germany { Biophysics of Genome Structure, Institute for Pharmacy and Molecular Biotechnology and KirchhoffInstitute for Physics, University Heidelberg, Heidelberg, Germany { Munich Center for Integrated Protein Science, Munich, Germany 1 Present address: CRT-RIV—ITAV (Universite´ de Toulouse—UPS, INSA, CNRS, UMS), Toulouse Cedex 1, France International Review of Cell and Molecular Biology, Volume 282 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)82001-5

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2010 Elsevier Inc. All rights reserved.

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Abstract In this review we describe major contributions of light and electron microscopic approaches to the present understanding of functional nuclear architecture. The large gap of knowledge, which must still be bridged from the molecular level to the level of higher order structure, is emphasized by differences of currently discussed models of nuclear architecture. Molecular biological tools represent new means for the multicolor visualization of various nuclear components in living cells. New achievements offer the possibility to surpass the resolution limit of conventional light microscopy down to the nanometer scale and require improved bioinformatics tools able to handle the analysis of large amounts of data. In combination with the much higher resolution of electron microscopic methods, including ultrastructural cytochemistry, correlative microscopy of the same cells in their living and fixed state is the approach of choice to combine the advantages of different techniques. This will make possible future analyses of cell type- and species-specific differences of nuclear architecture in more detail and to put different models to critical tests. Key Words: Chromosome territory, Chromatin domain, Perichromatin region, Nuclear functions, Fluorescence microscopy, Light and electron microscopy, superresolution. ß 2010 Elsevier Inc.

1. Introduction Impressive progress could be witnessed during the last 10 years in the field of epigenetics. Based on multifaceted studies of covalent histone modifications and their interplay with DNA methylation sites, as well as histone variants and chromatin remodeling events, molecular biologists have attempted to decipher what may be called the chromatin language (Guil and Esteller, 2009; Jiang and Pugh, 2009; Mercer et al., 2009; Munshi et al., 2009; Strahl and Allis, 2000; Varga-Weisz and Becker, 2006). Understanding this language, its species-specific modifications and the mechanisms involved in its cell type-specific expression, has become a central part of ongoing efforts to understand genome and nuclear functions. In comparison with the widely used term histone code ( Jenuwein and Allis, 2001; Strahl and Allis, 2000), the term chromatin language emphasizes that histone modifications do not provide a code in the sense of DNA triplets coding for a certain amino acid, but must be read and understood in the context of other modifications of the chromatin environment. As the fertilized egg progresses through development and translates its information into a multitude of cell fates, one genome can generate many ‘‘epigenomes’’ (European Epigenome Network of Excellence: What is epigenetics? http://www. epigenome-noe.net/aboutus/epigenetics.php). Whereas researchers in

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Epigenomics Goal: understanding the functional integration of epigenetics and nuclear architecture Nuclear architecture

?

Epigenetics

Goal: decipherment of universally valid, speciesand cell type-specific rules of nuclear organization

Goal: decoding the chromatin language

Epigenomics = epigenetics + nuclear architecture

Figure 1.1 The authors’ view of epigenetics and epigenomics.

epigenetics attempt to understand the chromatin language, we argue that understanding the functional implications of a multitude of epigenomes will not be possible by epigenetic studies alone, but requires in-depth studies of nuclear architecture as well (Fig. 1.1). Nuclear architecture represents the highest level of structural order and information content of the epigenome. Detailed knowledge of the dynamic nuclear architecture is indispensable for understanding gene regulation and other nuclear functions (Bartova et al., 2008; Chuang and Belmont, 2007; Fakan and van Driel, 2007; Fraser and Bickmore, 2007; Gasser, 2002; Kosak and Groudine, 2004; Lanctot et al., 2007a; Misteli, 2007; Pederson and Singer, 2006; Rippe, 2007; Spector, 2003; Taddei et al., 2004; Takizawa et al., 2008; Trinkle-Mulcahy and Lamond, 2008). Notably, the possible extent of functionally important cell type- and species-specific differences is still unknown. Knowledge of evolutionarily conserved structural principles and species-specific peculiarities of the nuclear architecture is still in an unsatisfactory state. Although it is now well established that changes of nuclear phenotypes, including changes of higher order chromatin arrangements, take place during differentiation of somatic cell types within a developing organism, the functional implications of such changes are not known. Nor do we know which differences of the nuclear architecture may play an important role in the deregulation of genes in cancer cells (Ye et al., 2007). No consensus has been reached with regard to the common principles of nuclear architecture shared by all eukaryotes. Contradictory models of nuclear architecture (discussed below) are a reflection of this fact. Repositioning of genes, from repressive to transcriptionally favorable nuclear compartments and vice versa, proposed by some of these models apparently correlate with changes of transcriptional activities. In addition, we are confronted with the task to explore the whole range of cell type- and

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species-specific differences and understand their functional implications. Attempts to analyze the extent of functionally important Brownian or directed chromatin movements are still in their very beginning, not to speak of the mechanisms which assure that any nucleus in an organism displays the correct higher order structure at the right place and the right time. Within the size limits of this review a full coverage of the present state of research in nuclear architecture was not possible. We refer readers to an addendum, where we provide a few pertinent references to topics excluded from further consideration. Topics excluded from this review include the nucleosome (Olins and Olins, 2003; Woodcock, 2006) and its role in epigenetic gene regulation, insulators, and boundaries that delimit differentially regulated loci (Capelson and Corces, 2004; Ishii and Laemmli, 2003; Lunyak, 2008), the disputed concept of nuclear matrix and higher order chromatin organization (Berezney and Coffey, 1974; Bode et al., 2003; Elcock and Bridger, 2008; Hancock, 2000; Kaufmann et al., 1986; Malyavantham et al., 2008; Pederson, 2000; Razin et al., 2007; Zink et al., 2004), higher order chromatin structures achieved by chromatin self-assembly (Albiez et al., 2006; Misteli, 2001; Poirier and Marko, 2002), structure and function of the nucleolus (Boisvert et al., 2007; Hernandez-Verdun, 2006; McKeown and Shaw, 2009), the nuclear envelope, its pore complexes, and interaction with chromatin (Akhtar and Gasser, 2007; Kalverda et al., 2008; Maco et al., 2006; Prunuske and Ullman, 2006), composition and molecular biology of nuclear bodies and inclusions (Bernardi and Pandolfi, 2007; Gall, 2003; Morris, 2008; von Mikecz, 2009), interchromatin granules or splicing speckles (Fakan, 2004; Lamond and Spector, 2003; Thiry, 1995a), topography and functional roles of telomeres (Gasser et al., 2004; Hediger and Gasser, 2002). We also excluded nuclear architecture and chromosome dynamics in meiotic cells from this review (Kleckner, 2006; Scherthan et al., 2007; Vazquez-Nin et al., 2003), as well as topics with established or potential clinical applications, such as laminopathies (Dechat et al., 2008; Goldman et al., 2005), nuclear architecture in senescent cells (Funayama and Ishikawa, 2007; Malatesta et al., 2003; Narita, 2007; Oberdoerffer and Sinclair, 2007) and cancer cells (Londono-Vallejo, 2008; Zaidi et al., 2007; Zink et al., 2004). We attempt to provide an overview of microscopic studies exploring the functional nuclear architecture. In the first part we summarize what is known, and more important, what is still not known about the in situ topography of chromatin and various other nucleoplasmic components. We point out the necessity to investigate nuclear architecture at the single cell level, including the various compactions and lengths of chromatin fibers or loops and the dynamics of higher order chromatin arrangements. In the second part it is our goal to provide molecular biologists with the background necessary to better understand the complex world of quantitative microscopy and image analysis. Molecular biologists may be at a loss to judge

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the possibilities and limitations of microscopic approaches and consequently the reliability of quantitative microscopic data (Ronneberger et al., 2008). We also review recent breakthroughs in fluorescence microscopy (FM), which have opened access to the observation of structures at nanoscale dimensions (< 100 nm), and discuss how these new approaches can be most fruitfully combined with the superior resolution of electron microscopy (EM). In this context we argue for the necessity of correlative microscopic studies of nuclear architecture following the same cell, first in the living and then in the fixed state, from the level of conventional and advanced 3D (space) and 4D (space–time) FM to the EM level. Advanced microscopic studies of nuclear architecture would be impossible without uncounted probes for the visualization of specific chromatin and nonchromatin domains in a multicolor format. Examples include DNA probes of different complexity for the visualization of chromatin structure and order at all levels ranging from entire chromosome territories (CTs) to single genes by fluorescence in situ hybridization (FISH) under conditions which conserve the spatial nuclear arrangement as much as possible at this level of resolution (Cremer and Cremer, 2001, 2010; Solovei et al., 2002). Three-dimensional (3D) RNA FISH has allowed not only to identify patterns of the global nuclear topography of transcription but also to determine the nuclear positions of individual transcriptionally active genes (Capodieci et al., 2005). The possibility to express specific nuclear proteins tagged with fluorescent proteins in combination with fluorescence recovery after photobleaching (FRAP) technology has revolutionized our insights into the dynamics of protein movements in the cell nucleus (Lippincott-Schwartz et al., 2003; Misteli, 2001). Expression of nuclear proteins tagged with fluorescent proteins and the construction of transgenes with DNA recognition sites that bind to specific fluorescently labeled proteins has provided a boost for studies of higher order chromatin arrangements and gene position in nuclei of live cells (Gerlich et al., 2003; Janicki et al., 2004; Kanda et al., 1998; Robinett et al., 1996; Walter et al., 2003b). Recently, Rothbauer et al. (2006) generated fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells and represent a new targeting tool (Rothbauer et al., 2006). The generation of many more in vivo probes for multicolor live-cell imaging of various nuclear structures will be one of the most important needs of microscopists from molecular biologists in the near future. Considering the necessity that such probes should not interfere with nuclear functions in unexpected ways, this task is a very demanding one. Novel molecular methods have made possible the genome-wide mapping of protein–DNA and DNA–DNA interactions. Although a detailed discussion of these techniques is beyond the scope of this review, we wish to emphasize their unique possibilities for studies of the nuclear

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architecture and complementarity to microscopic approaches. An approach, which allows the construction of genome-wide maps of DNA sequences interacting in vivo with DNA binding proteins, was introduced by van Steensel and Henikoff (2000). This method is based on the expression of a fusion protein, which consists of the protein of interest and the DNA adenine methyltransferase (Dam). DNA sequences carrying methylated adenines in the vicinity of the protein of interest are amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays (Vogel et al., 2007). A recent breakthrough, achieved with this elegant method, was the genome-wide characterization of DNA sequences interacting with the lamina underpinning the nuclear envelope in Drosophila melanogaster and human cells (Guelen et al., 2008; Pickersgill et al., 2006). Another approach with a great potential for nuclear architecture studies, called ‘‘chromosome conformation capture’’ (3C), was introduced by Dekker et al. (2002). The method is based on in situ formaldehyde crosslinking of DNA–DNA interactions. The original 3C approach has prompted numerous improvements to determine nonrandom spatial interactions between CTs at the level of specific DNA segments in cis (sequences within the same CT) and trans (sequences from different CTs) (Babu et al., 2008; Dekker, 2008; Dostie et al., 2007; Hu et al., 2008; Lin et al., 2009; Ling et al., 2006; Lomvardas et al., 2006; Noordermeer et al., 2008; Nunez et al., 2009; Osborne et al., 2007; Sexton et al., 2009; Simonis and de Laat, 2008; Simonis et al., 2006; Tiwari et al., 2008; Williams and Flavell, 2008; Zhao et al., 2006). The combination of circular chromosome conformation capture (4C) with DNA microarrays (Go¨ndo¨r et al., 2008; Schoenfelder et al., 2009) or massively parallel sequencing (Lieberman-Aiden et al., 2009) has allowed for the first time mapping of DNA–DNA interactions in cis and trans at a genome-wide level. Although DNA interactions in cis were found to abound, significant interactions in trans were detected in agreement with other studies cited above. This wealth of data, often substantiated by 3D FISH assays, has provided strong support for long-range interactions in cis, that is, between genes located many megabases (Mbs) apart on the same chromosomes, or trans, that is, between genes located on different chromosomes. To date, numerous spatial interactions of CTs were reported in cis and trans (Babu et al., 2008; Ling et al., 2006; Lomvardas et al., 2006; Noordermeer et al., 2008). Notably, 3C assays provide ‘‘high-throughput’’ possibilities to screen epigenomes globally for interactions in cis and trans. Their very principle, however, prevents their application at the single cell level. Present assays typically require some 10–20 million cells to screen for nonrandom interactions between DNA sequences. In cultured cell populations and in tissues consisting of a variety of different cell types, 3C assays cannot pinpoint statistically significant increases of specific DNA interactions to a specific cell type. Furthermore, these assays are complex and demand strict controls

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in order to escape the danger of false positive or negative findings (Dekker, 2006; Simonis et al., 2007). High-throughput assays and microscopic approaches are complementary (Simonis and de Laat, 2008). Quantitative microscopic approaches are needed to validate the findings of 3C assays and they are indispensable to determine the cell types in complex tissues, which show chromatin interactions in cis and trans. More importantly, microscopic approaches are the only way to reveal the entire structure of nuclear components and to determine their topography with respect to each other. Microscopic techniques have now been developed to the point, where they are capable to resolve interactions at the molecular level in the living cell.

2. Historical Background Since the late 19th century (Boveri, 1888; Rabl, 1885), an uncounted number of microscopic studies have appeared on numerous aspects of nuclear structure, yet methodology to investigate nuclear architecture at high resolution and in three dimensions became only available during the second half of the 20th century. Starting in the 1960s, first insights into the functional compartmentalization of cell nuclei were achieved by transmission electron microscopy (TEM) of nuclei (Fakan and Bernhard, 1971; Monneron and Bernhard, 1969; Olins et al., 1983; Smetana and Hermansky, 1963), followed during the 1980s by advanced 3D light microscopy methods (Agard and Sedat, 1983; Belmont et al., 1987; Popp et al., 1990). The basic repeating unit of chromatin, known since 1975 as the nucleosome (Oudet et al., 1975), was described in the 1970s (Kornberg and Thomas, 1974; Olins and Olins, 1974; Woodcock et al., 1976a,b). Studies of chromosome arrangements at this time were restricted to mitotic cells except for a few special cases (Comings, 1980). We focus this brief historical account on the discovery of CTs described in detail elsewhere (Cremer and Cremer, 2006a,b). To our knowledge, Carl Rabl (1885) and Eduard Strasburger (1905) were the first scientists who argued for a territorial organization of chromosomes in animal and plant nuclei, respectively. Theodor Boveri introduced this term in a seminal publication from 1909 arguing that each chromosome retains its individuality during interphase and occupies a distinct part of the nuclear space (Boveri, 1909). Notably, this hypothesis fell into oblivion during the 1950s–1980s, when only few researchers still adhered to this seemingly outdated concept, while most seemed to be content with the idea that chromatin fibers and loops intermingle in the nuclear space like a dish of spaghetti. Based on chromatin staining with a modified Giemsa banding technique, Stack et al. (1977) described CTs in nuclei from Allium cepa root tips and a Chinese hamster cell line, whereas the Cremer group obtained

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evidence for CTs in nuclei of diploid Chinese hamster cells with the help of laser–UV-microirradiation experiments (l ¼ 257 nm) (Cremer et al., 1982a,b, 1984; Zorn et al., 1976, 1979). These experiments were based on the following rationale (Cremer and Cremer, 2006b; Meaburn and Misteli, 2007): (1) The microbeam was used to produce UV-damaged DNA within a small part of the nucleus (about 5% of the total nuclear area). (2) The damaged DNA was traced from interphase to mitosis and identified on metaphase chromosomes. (3) Decisively different patterns of damage distribution on metaphase chromosomes were predicted for a territorial and a nonterritorial organization of interphase chromosomes (Cremer et al., 1982a). Two procedures were followed for the in situ detection of damaged DNA: Cells microirradiated in G1 were pulse-labeled with 3H-thymidine. Incorporation of the radioactive compound into microirradiated DNA was obtained during excision repair of DNA photolesions. Alternatively, microirradiated DNA was visualized by indirect immunofluorescent staining with antibodies raised against UV-damaged DNA. The latter approach made it possible to follow chromatin microirradiated at any time during interphase (including S-phase) from interphase to mitosis (Cremer et al., 1980, 1984; Hens et al., 1983). As predicted for Boveri’s early CT hypothesis, the results demonstrated that only a few mitotic chromosomes showed heavily labeled segments (Rabl, 1885; Strasburger, 1905). These segments constituted the parts of neighboring CTs hit by the microbeam. In situ hybridization experiments made it possible to visualize CTs in mammalian interphase nuclei (Cremer et al., 1988, 1993; Lichter et al., 1988; Manuelidis, 1985a, 1990; Pinkel et al., 1988; Schardin et al., 1985). Since the 1990s, FISH combined with either epifluorescence microscopy (EFM) or confocal laser scanning fluorescence microscopy (CLSM) became the favorite approach for studies of higher order arrangements of CTs as well as individual genes (see below).

3. Models of Nuclear Architecture and Experimental Evidence 3.1. Current models The severe limitations in our present knowledge of nuclear architecture become obvious, when we consider current models in recent scientific publications and textbooks (Fig. 1.2) (Cremer and Cremer, 2006b). Presently discussed concepts of higher order chromatin organization differ with regard to the fractions of the nuclear space occupied by transcriptionally permissive euchromatin and transcriptionally silent facultative heterochromatin, as well as the size distribution (DNA content) and

C

A Nuclear envelope

1 Mb chromatin domains

Chromosome territory

Splicing speckle

Interchromatin compartment (IC)

Nucleolus

Perichromatin region

1 mm − Transcription CTs with IC invaginations

+ Transcription

Nuclear neighborhood Nuclear neighborhood for gene silencing for gene expression

D

B Chromosome territory

Gene A Splicing speckle

Gene B

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Gene C

Nucleolus

Nuclear envelope

Cell changes in response to signals

Movement to different nuclear neighborhoods: genes A and C become highly active and gene B becomes silenced in heterochromatin

Figure 1.2 Different models of chromatin organization and topology of gene expression. (A) The chromosome territory–interchromatin compartment (CT–IC) model (Cremer and Cremer, 2001, 2006b; Cremer et al., 2000) argues for the coexistence of highly folded CTs built up from chromatin domains and a nearly DNA-free IC expanding between these domains. The perichromatin region contains decondensed chromatin and provides the border zone between the rather compact interior of chromatin domains and the IC (Fakan, 2004). The PR is the major functional, nuclear subcompartment. DNA transcription, RNA splicing, as well as DNA replication and DNA repair take place within

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compaction levels of chromatin loops/fibres. The CT–IC (chromosome territory–interchromatin compartment) model argues that nuclei are built up from two principal components, CTs and the IC (Cremer and Cremer, 2001, 2010; Cremer et al., 2000). The territorial organization of interphase chromosomes is now generally accepted as a basic principle of nuclear organization in both animals (Cremer and Cremer, 2001) and plants (Berr et al., 2006; Pecinka et al., 2004; Shaw et al., 2002) and may even hold for single cell eukaryotes, such as budding and fission yeast (Bystricky et al., 2005; Molnar and Kleckner, 2008). In multicellular organisms studied to date, a large chromatin fraction appears to be organized as 1 Mb chromatin domains first detected in S-phase nuclei as replication foci, but later shown to be persistent higher order chromatin structures (Albiez et al., 2006; Jackson and Pombo, 1998; Visser and Aten, 1999). Larger chromatin clumps may be composed of clusters of 1 Mb domains. According to the CT–IC model, 1 Mb chromatin domains constitute a basic structure of CTs (Albiez et al., 2006; Berezney et al., 2005), but neither their ultrastructural organization nor the packaging of chromatin connecting these domains has been clarified to date (see below). The IC concept asserts an apparently DNA-free, contiguous space, which expands between the nuclear pores and the higher order chromatin network described above. The IC harbors nonchromatin nuclear domains such as interchromatin granules or splicing speckles as well as a variety of nuclear bodies (Albiez et al., 2006; Fakan, 2004; Verschure et al., 1999; Visser et al., 2000). The entire IC is separated from the more condensed interior of chromatin domains and/or higher order chromatin fibers by a layer of more decondensed chromatin and fibrogranular RNP constituents, termed the perichromatin region (PR) (Fakan and van Driel, 2007). Topographically, the PR, therefore, represents the utmost periphery of a given chromatin the PR (cf. Figs. 1.4 and 1.8). (B) The interchromatin network (ICN) model (Branco and Pombo, 2006; reproduced with permission) proposes that euchromatin is made up from chromatin fibers, which intermingle more or less homogeneously by constrained diffusion both in the interior of CTs and between neighboring CTs. Blue dots signify interchromosomal contacts maintained by tethering. (C) The giant loop field (GLF) model (Chubb and Bickmore, 2003) suggests that transcription occurs on giant chromatin loops which expand from the surface of CTs and form a field of intermingling loops. When transcription ceases, the giant loops collapse back onto condensed core domain of CTs, which is typically visualized by chromosome painting. (D) The longrange interaction (LRI) model (Figure 4-66/page 241; Alberts et al., 2008, reproduced with permission) shows the most extreme version of a giant loop model. Giant loops can be very long and expand throughout the whole nuclear space in order to carry genes located on them even to very remote nuclear sites. In this way several genes can congress within the same ‘‘nuclear neighborhood for gene expression’’ or expression hub (Kosak and Groudine, 2004) and be transcribed there in a coregulated manner. In addition, the LRI model suggests genes on very long giant loops can reach distant nuclear neighborhoods for gene silencing.

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domain. Functionally, we argue that it presents the essential subnuclear compartment for DNA replication and repair, transcription, and premRNA splicing (Fig. 1.2A). If one wishes to define the interchromatin space simply as any chromatinfree nuclear space, such a space is an implicit part of any model of higher order chromatin arrangements including the proposition that the nucleoplasm is filled by intermingling chromatin fibers. The ‘‘lattice’’ model of interphase chromatin proposed by Dehghani et al. (2005) suggests a lattice-like network of 10 and 30 nm fibers. This structure yields a porous organization of chromatin with fibers intermingling at the borders of neighboring CTs. In agreement with this claim, the interchromatin network (ICN) model (Branco and Pombo, 2006) predicts that chromatin fibers and loops intermingle in a rather uniform way, both in the interior of individual CTs and between differentially labeled neighboring CTs, making any distinction between the interior or periphery of distinct chromatin domains functionally meaningless (Fig. 1.2B). In this ICN, loops may expand from one CT to meet loops from another CT. The ICN model does not provide any argument for a special nuclear topography of transcriptionally active and silent compartments. Still, other models favor giant chromatin loops, which emanate from dense chromatin domains (Chubb and Bickmore, 2003; Fraser and Bickmore, 2007). The Bickmore group has argued that ‘‘the organization of chromosomes within the nucleus is probably somewhere in between the complete decondensation of chromatin fibers like spaghetti on a plate suggested 30 years ago and the model of a discrete territorial organization favored recently’’ (Mahy et al., 2002a) (Fig. 1.2C). For a short synonym in the following text, we name this model the GLF (giant loop field) model. An extreme case of a giant loop model was proposed in the fifth edition of the textbook Molecular Biology of the Cell (Alberts et al., 2008) (Fig. 1.2D). According to this model, which for brevity, we will refer to as the LRI (long-range interaction) model, giant loops expand throughout the nuclear space in order to carry genes located on them even to very remote sites across the whole nuclear space. Several genes can congress within the same ‘‘nuclear neighborhood for gene expression’’ or expression hub (Kosak and Groudine, 2004) and be transcribed there in a coregulated manner. In addition, this model suggests nuclear neighborhoods for gene silencing in order to carry genes to such repressive nuclear compartments. The lack of quantitative rigor has limited the usefulness of the models described above. To overcome this situation, chromatin polymer models have been developed, which make experimentally testable, quantitative predictions about functionally important features of the nuclear architecture, such as the expected size distribution of chromatin loops and chromatin compaction levels. Attempts have been made to explain the structure and nuclear arrangements of chromatin by random walk polymer models assuming a looped chromatin organization. The size of the loops predicted by different models

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ranges widely. The random-walk/giant-loop model has argued for a randomwalk backbone of an ill-defined nature to which chromatin loops of 3 Mb are attached (van den Engh et al., 1992). The multiloop-subcompartment (MLS) model has proposed rosette-like structures consisting of multiple 120-kb loops (Munkel and Langowski, 1998; Munkel et al., 1999), which provide a possible explanation for the organization of replication foci/1 Mb chromatin domains (Cremer and Cremer, 2001). A recent chromatin polymer model has assumed a broad range of loop sizes (Mateos-Langerak et al., 2009). All chromatin polymer models need to explain that chromatin loops representing an individual chromosome are spatially constrained to a confined nuclear subvolume, the CT.

3.2. Experimental evidence 3.2.1. Chromosome territories and interchromatin spaces Whereas CTs are now fully accepted as a basic feature of nuclear organization, the question of an interchromatin space or more specifically of an IC as predicted by the CT–IC model (see above; Cremer and Cremer, 2001, 2010; Cremer et al., 2000) has remained controversial. An interchromatin space was first described in EM studies in the 1960s (Monneron and Bernhard 1969). After the (re)discovery of CTs the existence of a network-like space, called the interchromosomal domain (ICD), was suggested expanding mainly around CTs with little penetration into the CT interior (Cremer et al., 1993; Zirbel et al., 1993). Supposedly, genes were preferentially transcribed in a region of decondensed chromatin delineating CT surfaces and RNA transcripts were directly released in the ICD compartment. This concept was later supported by a series of studies from the Lichter group (Bridger et al., 1998, 2005; Gorisch et al., 2003, 2005; Reichenzeller et al., 2000; Richter et al., 2005). Accumulating evidence for genes transcribed both outside and in the interior of CTs (Mahy et al., 2002b; Verschure et al., 1999) is consistent with electron microscopic evidence for a network-like DNA-free space both outside and inside CTs (Visser et al., 2000). Notably, the conventional staining of ultrathin sections with uranyl acetate and lead citrate is unspecific. Accordingly, DNA, RNA, and proteins cannot be discriminate. Thus, it can be difficult, if not impossible, to distinguish a rather DNA-free IC full with RNPs and protein complexes from neighboring chromatin clusters. To do so it is necessary to combine EM studies with DNA-specific staining procedures. Indeed, such EM studies have provided strong evidence in favor of an interchromatin space which expands between chromatin clusters. Figure 1.3 shows the interchromatin space and the DNA distribution in a 2D micrograph and 3D EM reconstruction of a rat hepatocyte nucleus following specific DNA staining (Rouquette et al., 2009) (cf. Fig. 1.4). This 3D reconstruction is in full agreement with expectation of the CT–IC model (Fig. 1.2A) but not with

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A

B D

1 mm

4.5 mm

Figure 1.3 3D reconstruction of a rat liver cell nucleus imaged by serial block-facescanning electron microscopy (SBF-SEM). SBF-SEM was applied after a selective preembedding staining for DNA (for more details see Rouquette et al., 2009). (A) A single SEM image extracted from an image stack demonstrates the strongly inhomogeneous distribution of DNA in higher order chromatin clusters and the wide interchromatin space expanding between them. (B) A reconstruction based on 90 consecutive images representing the major part of the nucleus (4.5 mm in z direction) gives the impression that a very large part of the nuclear volume is more or less homogenously filled with chromatin creating the false impression that the distinctly separate interchromatin (see Fig. 1.2A, CT–IC model) space expanding between chromatin clusters with wide channels and lacunas may not exist at all (see Fig. 1.2B, ICN model).

the ICN model (Fig. 1.2B). It was based on 170 sequential images obtained with a scanning electron microscope. After each image a slice of 50 nm was removed with a built-in ultramicrotome (Denk and Horstmann, 2004). The interchromatin space in rat hepatocyte nuclei occupied about 65% of the total nuclear volume, whereas endothelial cell nuclei in the same tissue occupied only about 40%. These values agree with 3D EM reconstructions of serial ultrathin sections of interphase nuclei indicating about half of nuclear space filled by chromatin (Esquivel et al., 1989; Lopez-Velazquez et al., 1996). It should be noted that values for the relative space taken by chromatin and the interchromatin space, respectively, do not suffice to distinguish between the CT–IC and the ICN models, although the actual chromatin distribution predicted by both models is decisively different (Fig. 1.2A and B). Based on this evidence, the hypothetical CT structure suggested by the CT–IC model may best be compared to a sponge of chromatin permeated by intraterritorial IC channels (cf. also Fig. 3 in Visser et al., 2000), as well as 3D reconstructions of CTs based on light optical serial sections (cf. Fig. 24 in Cremer and Cremer, 2006b). Individual CTs form an interconnected network of compact higher order chromatin domains and are closely associated with a second contiguous 3D spatial network observed both at the FM and

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EM levels and called the IC or interchromatin space (Albiez et al., 2006; Visser et al., 2000). EM studies demonstrating chromatin clusters can be integrated with other EM studies arguing for the packaging of a large proportion of mammalian chromatin into 60–130 nm ‘‘chromonema’’ fibers (Belmont and Bruce, 1994). In spite of circumstantial evidence for a nonrandom, dynamic organization of CTs described below, studies to define cell typeand species-specific differences between ultrastructural features of CTs are still in their beginning (Rego et al., 2008). In stark contrast to the CT–IC model, the ICN model argues for high levels of intermingling between chromatin loops inside CTs and between CTs. Whereas evidence that space between chromatin adds up to about half of the total nuclear space fits with both the CT–IC model and the ICN model, one should be aware of the major differences of chromatin organization predicted by both models. The ICN model was proposed on the basis of FISH experiments with chromosome painting probes to thick cryosections (140–180 nm) from cell nuclei (Branco and Pombo, 2006; Pombo, 2007); the hybridized sections were first examined by FM and subsequently by TEM employing a detection scheme, where the hybridization sites of two differentially labeled chromosome paint probes were recognized with colloidal gold particles of different size. Occasional intermingling of neighboring CTs is consistent with a previously reported observation on in vivo labeled cells describing direct contacts between a labeled chromatin domain/territory and its unlabeled neighbor (Visser et al., 2000). In control experiments, Branco and Pombo (2006) performed EM on nuclei where histone H2B was indirectly immunolabeled with gold particles, and the positions of gold grains did not significantly change when cryosections were studied before and after mock FISH. Examination of the published electron micrographs does not, however, allow a morphological orientation in the section with regard to chromatin domains and other nuclear structures. Accordingly, the level of structural preservation in these cryosections remains doubtful. Nevertheless, one has an impression of a chromatin-poor/-free interchromatin space, which expands between chromatin domains (see Fig. 1J and K in Branco and Pombo, 2006). In order to resolve conflicting opinions about intermingling chromatin loops, it is necessary to provide quantitative data about the numbers, length and compaction levels of such fibers, and their 3D distribution. 3.2.2. Chromatin loops: How many, how long, and how compacted? Although numerous studies have presented evidence for 11 and 30-nm-thick chromatin fibers as well as higher order chromatin configurations (Belmont et al., 1999; Gilbert et al., 2004, 2005), chromatin configurations above the 11-nm fiber have remained controversial (Sapojnikova et al., 2009). Recent cryoelectron microscopic studies carried out on vitrified sections of in situ interphase nuclei of different cultured

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IS

C

C

Figure 1.4 Vitrified ultrathin section of a PtK2 cell nucleus viewed by cryoelectron microscopy. The cells were fixed as close as possible to their native state by highpressure freezing and the section shows only its inherent contrast, in the total absence of any chemical fixative or contrasting agent (Bouchet-Marquis et al., 2006). The dotted line indicates the border of a condensed chromatin domain (C) from which the PR consisting of fibrogranular material emerges. The fine stippled line roughly shows the transition region between the PR and the interchromatin space (IS). Arrows indicate ice crystals contaminating the cryosection. Bar represents 200 nm. Reprinted from Fakan and van Driel (2007), with permission from Elsevier.

mammalian cells (Bouchet-Marquis et al., 2006) (Fig. 1.4) or HeLa cell mitotic chromosomes (Eltsov et al., 2008) were unable to reveal 30-nm chromatin fibers. The variation in length of fibers/loops of different compaction levels and the fraction of the genome packaged in this way may strongly vary with the global transcription level of a given cell at different functional states. Such data are still lacking for most cell types. Heitz (1928) originally introduced the terms heterochromatin and euchromatin to discriminate between genetically inactive and active chromatin. While constitutive heterochromatin typically lacks genes and is ubiquitously present as compact chromatin in all cell types of an organism, such as pericentromeric heterochromatin, the structural and/or functional definitions of facultative heterochromatin and euchromatin have remained ambiguous to date. The rapidly evolving field of epigenetics has provided for the first time possibilities to characterize functionally different types of chromatin at the molecular level (Berger, 2007; Brink et al., 2006; Trojer and Reinberg, 2007). Facultative heterochromatin comprises silent genes, which are inactive only under specific circumstances. The inactive X-chromosome in female somatic cells of mammals provides

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a case in point. Arguably, facultative heterochromatin has a ‘‘closed’’ configuration in contrast to the ‘‘open’’ configuration of euchromatin. An ‘‘open’’ chromatin context, however, does not imply that all genes present in such a configuration are actually transcribed. Structural differences of active and inactive chromatin in the living cell are still surprisingly illdefined. Transcriptionally poised or active chromatin may show a wide range of different compaction states. Thus, the notion of compact chromatin is not necessarily equivalent with the notion of heterochromatin. Despite this confusion there is an agreement that the relative amounts of active and inactive chromatin are developmentally regulated and can differ strongly in different cell types and different functional contexts. Whereas nuclei with a low transcription level, such as those in nonstimulated lymphocytes of the peripheral blood, typically show large blocks of condensed chromatin, nuclei with a high transcription rate, such as nuclei in PHAstimulated lymphocytes, often exhibit highly fragmented condensed chromatin domains (Pompidou et al., 1984). Several groups reported chromatin loops carrying specific clusters of genes expanding up to several micrometers away from the surface of their home CTs (Mahy et al., 2002a; Ragoczy et al., 2003; Volpi et al., 2000; Williams et al., 2002). Such an extrusion of a gene locus from a CT is not necessarily indicative of transcriptional activity, but also can reflect a poised state for activation (Ragoczy et al., 2003). Notably, the compaction level of one such giant loop studied in detail was about one order of magnitude higher than that of an extended 30-nm-thick chromatin loop (Albiez et al., 2006) (Fig. 1.5). Evidence for large numbers of giant loops expanding through major parts of the nuclear space as suggested by the LRI model (Fig. 1.2D) is meager. As a caveat, one needs to take into account that the sensitivity of chromosome painting experiments does not suffice to detect giant loops. 3D FISH experiments can only detect such loops and their true extension in the nuclear space with contig probes delineating the entire loop in question (Fig. 1.5). Both live-cell experiments and studies of fixed cell nuclei with TEM have supported the concept that most chromatin is compacted as focal chromatin domains with compaction levels above those of extended 30 nm chromatin fibers (Albiez et al., 2006). In live-cell experiments it was further demonstrated that the structural reorganization of CTs into mitotic chromosomes depends on the increased local compaction of chromatin—not the retraction of many giant loops expanding through a major part of the nucleus (Walter et al., 2003b). Again the limited sensitivity of present methods may have prevented the detection of occasional giant loops. Despite a considerable amount of information on the arrangement of CTs, their chromatin organization is not yet understood at the ultrastructural level. The particularly compact state of mitotic chromosomes makes it also extremely difficult to unravel their higher order

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tel 0 Mb

11 p15.5 1 Mb

2 Mb

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Figure 1.5 3D-FISH of 15 BACs spanning a gene-dense region on 11p15.5 (cf. Fig. 1.2C). Human fibroblast nucleus (stained with TOPRO-3; false colored in gray) after multicolor 3D FISH of the two HSA 11 territories (green) together with a gene-dense 2.35 Mbs region from HSA 11p15.5. Fifteen BACs were used for the delineation of this region in false colors: red for the most telomeric, blue for the most centromeric BAC, and yellow for 13 BACs covering the intermediate region as a contig except for 350 kb in the middle (not shown). The nuclear shape is represented by a maximum Z projection of TOPRO-3 sections. The 3D reconstruction of the genedense region reveals a finger-like chromatin protrusion with a compaction factor of  1:300 expanding from CT 11 (cf. Fig. 1 in Albiez et al., 2006).

structure (Belmont, 2006; Falconi et al., 2006; Wanner and Formanek, 2000). At present we do not know in sufficient detail the structural transformation of CTs into mitotic chromosomes. Evidence that the positions of genes harbored by a CT can change during activation or silencing of genes raises the question whether functionally relevant, positional information provided by a specific topography of genes in a given CT is still reflected by the 3D organization of the respective mitotic chromosome or whether such information is lost during mitosis and thus must be fully restored during the next interphase, most likely during early G1.

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3.2.3. Nonrandom arrangements of chromosome territories and chromatin domains Two cases of nonrandom higher order arrangements of CTs and subchromosomal regions have to be distinguished. Nonrandom radial arrangements describe the preferred location of specific chromatin structures, such as CTs, chromosomal subregions and genes, with respect to their 3D distance from the nuclear center or from the nuclear envelope. Nonrandom neighborhood arrangements reflect the proximity/clustering of such structures to an extent that cannot be explained as a consequence of a nonrandom radial organization. A search for molecular mechanism(s) involved in dynamic changes of higher order chromatin arrangements must be based on a well documented and fully reliable descriptive analysis of these changes. During evolution nonrandom proximity patterns of CTs, specific chromatin segments or genes may have been established primarily for nonfunctional reasons such as geometrical constraints. Later evolution, the old tinkerer, to use Francois Jacob’s famous expression, may have exploited such opportunities leading step by step to a preference of functionally advantageous proximity patterns between distinct chromatin domains and other nuclear structures. In most mammalian cell types studied to date, nonrandom radial CT positions were correlated with gene density and to some extent with chromosome size. In spherical cell nuclei, such as lymphocyte nuclei, positions of gene-poor CTs were typically found closer to the nuclear envelope than the positions of gene-dense CTs (Cremer et al., 2001, 2003; Croft et al., 1999). In flat ellipsoidal nuclei, such as nuclei in cultured fibroblasts, a chromosome size correlated distribution was found as the predominant feature (Cremer et al., 2001; Sun et al., 2000) although gene density-dependent features of CT positioning were also detected (Bolzer et al., 2005; Neusser et al., 2007). Notably, a study of bovine embryos generated by in vitro fertilization (IVF) revealed that this positioning is not present in pronuclei and blastomere nuclei during the first cell cycles but takes place only during major genome activation (Koehler et al., 2009). In murine cells lacking full-length lamin B1 or defective in processing its CAAX anchor positions of gene-dense and genepoor CTs are affected (Malhas et al., 2007). In case of CTs, which are built from gene-poor and gene-dense segments, the latter were typically observed in a more interior position (Neusser et al., 2007). Across a wide evolutionary spectrum, ranging from primates and other mammalian cells (Bolzer et al., 2005; Grasser et al., 2008; Kupper et al., 2007; Mayer et al., 2005; Tanabe et al., 2002) to birds (Habermann et al., 2001), hydra (Alexandrova et al., 2003), plants (Mayr et al., 2003), and single cell eukaryotes (Postberg et al., 2005), a considerable amount of dense chromatin/heterochromatin is situated at the nuclear periphery, while other dense chromatin/heterochromatin surrounds the nucleoli as well as the internal nuclear regions outside nucleoli.

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In contrast, euchromatin expands toward the nuclear interior together with dispersed interior clumps of condensed chromatin/heterochromatin. We refer to this organization as the conventional type of nuclear architecture. Possible functional reasons for this conventional architecture are unknown. A recent study of the mammalian retina (Solovei et al., 2009) has demonstrated that the nuclear architecture of rod cell nuclei is inverted in mammals adapted to low light conditions, while rod cell nuclei of mammals adapted to a diurnal life style possess the conventional architecture. In nuclei with the inverted architecture, all heterochromatin locates in the nuclear interior, while all euchromatin is located at the nuclear periphery. The inverted pattern forms by remodeling of the conventional one during postmitotic, terminal differentiation of rods. So far, the inverted nuclear architecture of rod cell nuclei from diurnal retinas seems to be a unique finding, indicating an adaptation of this nuclear architecture to the biophysical requirements of an effective channeling of photons toward the photoreceptors. The conventional architecture of eukaryotic nuclei may have prevailed in most cells during evolution, because it provides opportunities for more flexible chromosome arrangements facilitating a cell typespecific positional regulation of nuclear functions (Solovei et al., 2009). Nonrandom neighborhood arrangements or proximity patterns between certain nonhomologous CTs were also discovered (Parada et al., 2002; Roix et al., 2003). This proximity, however, was typically detected in a minority of nuclei, but never in all nuclei of a given cell population (Parada et al., 2004). This finding argues either for the possibility that nonrandom CT neighborhood arrangements are functionally required only in a subset of nuclei (Parada et al., 2003) or for the transient nature of a given proximity pattern established only at certain periods during the cell cycle or at certain stages of postmitotic cell differentiation. Importantly, preferred CT arrangements seem to be probabilistic, that is, these arrangements occur more often than expected in case of a purely random arrangement, but they are not deterministic in the sense that all nuclei in a well-defined population of cells would reveal exactly the same arrangement (Zeitz et al., 2009). The analysis of a fixed cell population by chromosome painting resembles a snapshot of a group of individuals. Additional information on the dynamic behavior of the individuals is required to decide whether certain individuals already met before or will meet after the snapshot was taken. This can either be achieved by taking many snapshots of fixed samples at different time point or preferably by the observation of living cells. Numerous studies demonstrated cell type-specific differences of centromere arrangements (Billia et al., 1992; Borden and Manuelidis, 1988; Brero et al., 2005; Hu et al., 2008; Manuelidis, 1985b; Martou and De Boni, 2000; Park and De Boni, 1992; Solovei et al., 2004, 2009). Still, little is known about whether proximity patterns of whole CTs may change as well in correlation with changing functional states. Much more evidence for permanent or transient CT proximity patterns will be obtained when

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systematic live-cell studies of CT movements become possible in cycling and postmitotic cell types present in tissues. Another old and largely unsolved problem concerns the question of nonrandom associations between homologous CTs in somatic cell types. Such associations are a hallmark of somatic cell nuclei of D. melanogaster and other Diptera species (Fritsch et al., 2006; Hiraoka et al., 1993; Lowenstein et al., 2004), whereas in cell types of many other species the spatial order of homologous CTs appears quite variable (Bolzer et al., 2005; Cremer et al., 1982a, 1982b; Pecinka et al., 2004; Scherthan et al., 1996). This issue is, however, complicated by the possibility that somatic homologous associations or pairing may be restricted to certain chromosomes in certain cell types and functional states. A study from Arnoldus et al. (1989) provides a point in case. These authors performed in situ hybridization experiments with normal human brain tissues using one probe specific for the 1q12 heterochromatic block of HSA 1 and another for the centromere of HSA 7. In the cerebral and the cerebellar samples they found two HSA 7 centromere spots in 82% and 83%, respectively, of the nuclei. In situ hybridization with the chromosome 1 probe showed only one large spot in 82% of the cerebellum nuclei, yet two smaller spots in 69% of the cerebral nuclei. Subsequent studies reported examples for homologous associations of centromeric domains in normal and tumor cell nuclei (Henikoff, 1997; Iourov et al., 2006; Koeman et al., 2008; Vadakkan et al., 2006). In contrast to somatic pairing in Diptera, somatic associations were only found in a minority of the evaluated nuclear samples. Accordingly, such associations may either occur transiently or may be entirely lacking in most nuclei. In any case, compelling evidence for functional implications is lacking. Based on a study on coordinate gene regulation during mouse hematopoiesis (Kosak et al., 2007) suggested that proximity in the form of chromosomal gene distribution and homolog association may be the basis for organizing the genome for coordinate gene regulation during cellular differentiation. As a caveat, we note that CTs preferentially located close to the nuclear center show significantly smaller 3D distances and a higher frequency of homologous associations as compared to CTs located at the nuclear periphery. 3.2.4. Mobility of chromosome territories and chromatin domains Positional changes of CTs and chromatin domains as well as genes indicate the dynamic nature of higher order chromatin arrangements. Early experiments were performed with cells fixed at different times of development or after exposure to certain stimuli (De Boni, 1994). In a seminal investigation, Barr and Bertram (1949, 1951) described that upon electric stimulation of cat motor neurons, a ‘‘nucleolar satellite’’ (now known as the Barr body) moved from its usual position adjacent to the nucleolus, toward the nuclear membrane within a time course of several days. Borden and Manuelidis (1988) demonstrated a pronounced repositioning of the human X-territory

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in neurons of both males and females in electrophysiologically defined seizure foci, whereas other CTs (HSA 1, 9, and Y) showed more subtle positional changes. De Boni and coworkers reported the rearrangement of centromeric satellite DNA in hippocampal neurons exhibiting long-term potentiation (Billia et al., 1992) and in mouse dorsal root ganglion neurons exposed to the neurotransmitter GABA (Holowacz and De Boni, 1991). Live-cell experiments performed with cultured mammalian and Drosophila cells demonstrated locally constrained movements of subchromosomal domains (Abney et al., 1997; Bornfleth et al., 1999; Edelmann et al., 2001; Marshall et al., 1997). In HeLa cell nuclei, movements of chromatin domains appeared more pronounced during early G1 compared with the more constrained movements observed during G1 to late G2 (Edelmann et al., 2001; Walter et al., 2003b). Once established in early G1, the positioning of entire CTs appears stable for the whole interphase (Walter et al., 2003b; Zink et al., 1998). More studies are needed to distinguish chromatin movements, which are the result of random diffusion, from energy-dependent directed movements (Chuang and Belmont, 2007; Chuang et al., 2006; Levi et al., 2005). This question is particularly important with respect to reports of long-range chromatin movements involved in the congression of coregulated genes. Elegant studies with living budding yeast cells provided insights into the long-range compaction and flexibility of native chromatin (Bystricky et al., 2004). The question, to what extent a given proximity pattern established between CTs during a given cell cycle may be transmitted to the next one, has remained controversial (Gerlich et al., 2003; Kalmarova et al., 2008; Thomson et al., 2004; Walter et al., 2003b). Chromosome movements during prometaphase required to establish the metaphase plate can lead to major changes of side-by-side chromosome arrangements in the metaphase plate compared with the side-by-side arrangements of the respective CTs during the preceding interphase (H. Strickfaden, A. Zunhamer, D. Koehler, and T. Cremer, unpublished data). In summary, available evidence argues (a) for a pronounced cell-to-cell variation of CT neighborhood arrangements in cell types studied so far, (b) for the stability of a given CT neighborhood arrangement once established at the onset of interphase until the next prophase, and (c) for major, probabilistic changes of chromosomes during mitosis (Cvackova et al., 2009; Walter et al., 2003b). As a caveat it must be emphasized that this evidence was obtained from a few cell types cultured in vitro. 3.2.5. Nonrandom arrangements, repositioning, and nuclear convergence of genes Parameters correlated with nonrandom radial nuclear arrangements of chromosomal subregions and genes include regional gene density, transcriptional activity, and replication timing (Amrichova et al., 2003; Goetze et al.,

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2007; Grasser et al., 2008; Hepperger et al., 2008; Kupper et al., 2007; Mayer et al., 2005; Murmann et al., 2005; Neusser et al., 2007; Sadoni et al., 1999). Since gene density, transcriptional activity, and replication time of a given chromatin segment are correlated with each other (Caron et al., 2001), it is not easy to distinguish between the relative contributions of these parameters to radial gene positioning. Several parameters may have an influence and it seems necessary to examine this problem on a gene-by-gene basis rather than making global statements. Notably, radial positions of a given gene can change significantly between cell types, excluding the possibility that gene positioning is determined solely by the DNA sequence environment in which a given gene is embedded (Hepperger et al., 2008). Gene repositioning has emerged as an additional level of epigenetic gene regulation (Bartova and Kozubek, 2006; Lanctot et al., 2007a). During repositioning, genes move from a repressive nuclear environment into a neighboring compartment favorable for transcription or vice versa. For some genes their transcriptional silent or active state could be correlated with their positioning close to a domain of constitutive heterochromatin or away from it (Brown et al., 1999). In other cases the silent gene was positioned close to the nuclear envelope and the active one remote from it (Kosak et al., 2002). In multipotent progenitors and derived cell types from an in vitro model of murine hematopoiesis, Kosak et al. (2007) noted a significant clustering of coregulated genes, both with respect to their linear arrangement along mitotic chromosomes and the 3D arrangement of active genes in the nuclear interior. The prevalence of heterochromatin localized at the lamina and the observation of silenced genes in this peripheral nuclear subcompartment has supported the concept that the nuclear periphery is a largely repressive environment for transcription (Schneider and Grosschedl, 2007). Recent studies, however, support a more complex picture (Akhtar and Gasser, 2007; Deniaud and Bickmore, 2009; Taddei et al., 2006; Towbin et al., 2009). Several groups succeeded to tether specific chromatin segments to the nuclear envelope in living cells. They found that some genes were suppressed when closely associated with the envelope, but that others were not (Finlan et al., 2008; Kumaran and Spector, 2008; Reddy et al., 2008). Notably, A- and B-type lamins are organized into separate, but interacting, microdomains in the lamina and may contribute to form different microenvironments for gene regulation (Shimi et al., 2008). In a genome-wide survey of D. melanogaster Kc cells, van Steensel and coworkers detected 500 transcriptionally silent, late replicating genes that interact with B-type lamin (Pickersgill et al., 2006). Studies in yeast have shown the association of active genes with the nuclear pore complex (NPC) (Taddei, 2007). Considering the possible role of gene repositioning for gene regulation, one should not forget the importance of local fluctuations of unfolding chromatin with respect to the availability of genes to transcription machineries (Sato et al., 2004).

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Of particular interest are hints that a long-range spatial nuclear convergence of genes, which are located many megabases apart on the same chromosome or even on different chromosomes, might be involved in mechanisms of gene activation or silencing (Bartkuhn and Renkawitz, 2008; Zuckerkandl and Cavalli, 2007). This phenomenon has been referred to as ‘‘gene kissing’’ (Cavalli, 2007; Kioussis, 2005). As an early example, LaSalle and Lalande (1996) presented 3D FISH evidence for the transient spatial association of the AS/PWS loci during late S phase. These loci comprise the genes involved in two intensively studied imprinting disorders, the Angelman syndrome and the Prader–Willi syndrome. The authors argued that transient ‘‘kissing’’ between the two loci is required for maintaining opposite imprints in cycling cells. This specific case of ‘‘kissing,’’ however, could not be confirmed in a later study (Teller et al., 2007). Since expression of Hox genes located on different chromosomes is precisely regulated and synchronized during development, 3D distances between these genes were compared in cryosections of developing mouse embryos (Lanctot et al., 2007b). While chromatin decondensation and nuclear reorganization of the HoxB locus was noted upon induction of transcription (Chambeyron and Bickmore, 2004; Chambeyron et al., 2005), the frequency of ‘‘kissing’’ events was not significantly different in cells expressing a high proportion of the Hox clusters when compared to cells expressing none or only a few Hox genes. These results indicate that coregulation of the different Hox clusters is not associated with colocalization of the loci at a single regulatory compartment. In a 3D FISH study of Drosophila flies, Bantignies et al. (2003) demonstrated the involvement of Fab-7, a well-defined cellular memory module, in long-distance interactions in cis and trans. Since then 3C experiments, in part reconfirmed by 3D FISH experiments, demonstrated numerous ‘‘kissing events’’ between genes in cis and trans (Krueger and Osborne, 2006; Kurukuti et al., 2006; Lomvardas et al., 2006; Osborne and Eskiw, 2008; Osborne et al., 2004, 2007; Spilianakis et al., 2005; Tiwari et al., 2008; Williams and Flavell, 2008; Zhao et al., 2006). Evidence obtained by these experiments showed a percentage of colocalization between ‘‘kissing’’ genes large enough, say 10%, to consider a random occurrence quite unlikely. DNA interactions in cis abound a few megabases around a sequence chosen as a bait to search for interactions with other sequences (de Laat and Grosveld, 2007). In this case, constrained Brownian movements of chromatin domains are likely sufficient to enable a ‘‘kiss’’ (Bornfleth et al., 1999; Chuang and Belmont, 2007). Constrained Brownian movements may also suffice to reposition silent genes located in a repressive ‘‘heterochromatic’’ compartment into a neighboring transcriptionally favorable ‘‘euchromatic’’ compartment. Repositioning events demonstrated to date at light microscopic resolution may be just the tip of the ‘‘iceberg’’ of dynamic events taking place between decondensed chromatin in the PR and underlying

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condensed chromatin at submicroscopic resolution. The local chromatin environment may finally turn out to be much more dynamic than previously thought. More sophisticated multicolor 3D FISH and evaluation schemes are required to clarify the topography of these ‘‘kissing’’ events. As a case in point we consider an experiment with a sample of diploid cell nuclei, where two genes located on different CTs are visualized together with painting of the nonhomologous CTs in different colors. Such a scheme could show whether pairs of colocalized genes are typically located remote from these CTs on giant loops or whether colocalization requires direct contact between a given pair of CTs. The loops could be visualized simultaneously with contigs of BAC pools covering a few megabase pairs of the immediate neighborhood of these genes in a third color. In addition to the determination of the fraction of colocalized genes in an unbiased sample, it is important to measure also 3D distances between pairs of non-colocalized genes. Such measurements could reveal whether these pairs show a range of 3D distances compatible with the possibility of a ‘‘kissing’’ event brought about by constrained Brownian motions. In case that a very variable or even random distribution of 3D distances between non-colocalized pairs of genes would be discovered, we would assume that pairs of genes with large distances, for example >5 m, would be unlikely candidates for a transient ‘‘kiss’’ elicited by constrained Brownian chromatin motion. A recent study of primary cultures of human mammary epithelial cells and the human breast cancer cell line MCF7 describes rapid 17-estradiol (E2)-induced interactions in trans between the GREB1 and the TFF1 loci located on chromosomes 2 and 21, respectively (Hu et al., 2008). Notably, the authors emphasize that HSA 2 and 21 territories were independently localized before E2 treatment. A significantly increased frequency of TFF1: GREB1 interactions, however, could already be demonstrated 15 min after E2-treatment (about 20% vs. 5% at time 0) and this fraction increased to nearly 50% within 1 h. About half of the cells exhibited monoallelic interactions, the other half exhibited biallelic interactions in trans. Although HSA 2 and 21 territories became intimately associated in many cells, the frequency of E2-induced TFF1:GREB1 interactions was even higher than HSA 2 and 21 territory associations, suggesting the contribution of longdistance DNA looping in addition to movements of the entire territories toward each other. Movements resulting in TFF1:GREB1 ‘‘kissing’’ could be prevented by treatment of the cells with latrunculin, a drug that blocks actin polymerization, with Jasplakinolide that inhibits depolymerization of actin networks, by siRNA knockdown of nuclear Myosin-I and finally by microinjection of antibodies against Myosin-I or g-actin. Finally, Hu et al. (2008) noted that upon E2 treatment the interacting TFF1/GREB1 loci became intimately associated with SC35-positive speckles (or interchromatin granules) and speculated that these granules may function as hubs for

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gene networking in the nucleus. This speculation contradicts evidence, which argues that interchromatin granules are a DNA-free domain located in the IC (Thiry, 1995b). 3.2.6. Local chromatin dynamics of transcription: Role of the perichromatin region Like DNA replication, the nuclear topography of transcription sites was first investigated by methods of ultrastructural cytochemistry combined with EM autoradiography using tritiated uridine and later with immuno-EM and halogenated RNA precursors. Transcription sites have been demonstrated to occur, after labeling pulses as short as 2 min, predominantly in the PR (Fakan and Bernhard, 1971). This region is also the nuclear subcompartment where most factors involved in pre-mRNA/hnRNA formation accumulate, such as RNA Pol II, PolyA polymerase, and different other RNA processing factors (Cmarko et al., 1999; Fakan et al., 1984; Spector et al., 1991; Trentani et al., 2003) as well as DNA–RNA hybrids (Trentani et al., 2003). A special contrasting method for ultrathin sections allowed the visualization of ribonucleoprotein constituents in the PR, called perichromatin fibrils (Bernhard, 1969; Monneron and Bernhard, 1969). A perichromatin fibril represents the in situ form of a nucleoplasmic RNA transcript (Fakan, 1994, 2004; Fakan et al., 1976; Nash et al., 1975; Puvion and Puvion-Dutilleul, 1996) and also the site where cotranscriptional splicing obviously occurs (Biggiogera et al., 2008; Fakan, 2004). The predicted interplay between chromatin topography, chromatin dynamics, DNA transcription and replication differs starkly between the CT–IC model, and the ICN and GLF models. According to the CT–IC model, one expects the localization of active genes within the PR, while silent genes might be located in the condensed interior of chromatin domains. The alternative that silent genes are also located at the periphery of chromatin domains is worth considering. This hypothesis has gained support by a study performed with two different human cell lines and with rat liver tissue showing the preferential location of Polycomb group (PcG) proteins HPC2, HPH1, BMI1, and RING1 in the PR, whereas they were virtually absent from the interior of condensed chromatin (Cmarko et al., 2003). Despite the substantial evidence provided in this review in favor for the existence of the IC and the PR as two topographically neighboring and functionally interacting, yet structurally distinct nuclear compartments, this concept has not been taken into account in pertinent, recent reviews (Fedorova and Zink, 2008, 2009; Fraser and Bickmore, 2007; Go¨ndo¨r and Ohlsson, 2009; Nunez et al., 2009; Pai and Engelke, 2010; Pombo and Branco, 2007; Sexton et al., 2009; Sutherland and Bickmore, 2009). Improvements of RNA FISH technology have provided the possibility to study gene expression at the single cell level with high spatial and temporal

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resolution (Levsky et al., 2002). We restrict the following considerations to the dynamic topography of transcription (Sinha et al., 2008). Everybody agrees on one point: transcription caught in action is performed on a short piece of naked DNA which has entered a channel-like part of the transcribing RNA Pol II molecule (Kettenberger et al., 2003). Whether the transcription machinery moves along the DNA or whether the DNA is pulled through RNA Pol II within an immobile transcription machinery, or whether both possibilities may be realized in vivo, is not known. 3.2.7. Nuclear topography of transcription: Transcription factories The concept of transcription factories (TFs) provides a different interpretation of transcriptionally active compartments ( Jackson, 2005; Sexton et al., 2007; Sutherland and Bickmore, 2009). Immunocytochemistry using brominated RNA precursors combined with FM allowed to observe some 300–500 transcription sites as multiple tiny spots ( Jackson et al., 1993; Wansink et al., 1993), whereas Iborra et al. (1996) reported a spotted appearance of some 2100 RNA Pol II sites per nucleus (range 2000–4400). Jackson et al. (1993) coined the term TF, assuming that these foci represent nuclear machineries, where transcripts are both synthesized and processed. Subsequent studies reported that RNA FISH signals from various transcriptionally active genes were typically (90% or more) associated with RNA Pol II domains or factories (Osborne et al., 2004, 2007; Ragoczy et al., 2006). Based on the assumption that the total number of genes transcribed at a given time is considerably higher than the number of detectable sites of ongoing transcription, it was proposed that several genes can be simultaneously transcribed by a single TF (Fraser and Bickmore, 2007). The problem of estimating the number of genes transcribed in a given cell at a given time point has become more complex due to the discovery of transcriptional pulsing, that is, the fact that individual genes are turned on and off at irregular intervals (Chubb et al., 2006). Furthermore, RNA FISH signals from genes sparsely transcribed away from TFs might not be distinguishable from diffuse fluorescent background. In published nuclear images, TFs appear quite variable with regard to numbers, shapes, and sizes. It is not clear to what extent notable differences between images of TFs, provided by different studies, reflect specifics of different cell types or different methods of TF visualization (nascent RNA, RNA Pol II) and image recording. The size of TFs and the percentage found for associations between RNA FISH signals and RNA Pol II foci depends, of course, to some extent on the threshold used for signal segmentation. Yet, the discrepancies between FM studies describing large numbers of quite tiny spots (Iborra et al., 1996; Jackson et al., 1993) and others presenting smaller numbers of much larger TFs of irregular form and shape with diameters of several hundred nanometers up to the micrometer scale (Sexton et al., 2007) are conspicuous. It seems possible that TFs described by

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different authors either as accumulations of newly synthesized RNA or RNA Pol II foci represent either different domains or different components of the same domains. In conclusion, although TFs have been taken for granted as distinct nuclear domains (Mitchell and Fraser, 2008), we still lack compelling ultrastructural, cytochemical, and molecular evidence for their existence as higher order functional units and for their topographical relationships with chromatin. Accordingly, concepts about their function as units for the coregulated, simultaneous transcription of several genes also remain speculative to date (Fig. 1.6A). Similarly, presently available data do not provide compelling evidence for the nuclear topography of TFs. In the context of the ICN model (Fig. 1.2B), we would expect that TFs should be present wherever they are needed within the masses of intermingling chromatin loops, be it in the interior or periphery of intermingling CTs. The GLF model (Fig. 1.2C) argues for the preferential location of TFs within fields filled with giant loops expanding between core CTs and carrying transcriptionally active genes. When transcription ceases, these loops collapse back onto the condensed core of a CT (Chubb and Bickmore, 2003). Indeed, proponents of the TF concept have argued for the preferential location of these transcription machineries in the space between CTs filled with chromatin loops carrying active genes (Fig. 1.6B) (Fraser and Bickmore, 2007). In contrast, the CT–IC model proposes that sites of transcription are restricted to the PR with its width of about 100–200 nm (Fakan and van Driel, 2007) (Fig. 1.4). Accordingly, perichromatin fibrils still attached to DNA should be found everywhere within IC channels with a width <400 nm, while in wider IC channels or lacunas, nascent RNA should not be significantly detectable in the interior. Numerous EM studies cited above support this assumption. At the present state of knowledge, we cannot exclude that studies of a wider variety of cell types from different species, in particular cells with high demands of global transcriptional activity, may provide examples where many chromatin loops expand from compact chromatin domains beyond the typical 100–200 nm width of the PR. In the case that intensive comparative studies fail to provide such examples, the functional importance of the PR as the nuclear subcompartment for transcription would be further strengthened. 3.2.8. Routes of mRNA export Whereas cotranscriptional splicing of nascent RNA present in perichromatin fibrils seems well documented, further possible modifications of these fibrils and routes to nuclear pores are still unsolved questions. Obviously, only a fraction of perichromatin fibrils contains RNA messages needed for protein synthesis in the cytoplasm, while others may contain RNAs which are retained and may serve regulatory processes in the cell nucleus. Many

A LCR transcript Inactive gene 3⬘ enhancer intergenic transcript

3⬘ enhancer elements

Boundary terminator

Intergenic transcript

LCR elements

Chromatin loop emerges

Bidirectional noncoding RNA (intergenic) promoter

Antisense intergenic transcript

Potentiated gene with distal 3⬘ enhancer elements

B

Chromosome territory

Cis and trans coassociation

Cis interaction/ trans interaction Speckle Transcription factory

Chromatin loop

Figure 1.6 Transcription factories. (A) Model of a transcription factory showing transcribed chromatin loops simultaneously moving through the factory at different sites (adapted by permission from Macmillan Publishers Ltd: Chakalova et al., 2005, copyright 2005). (B) Different types of gene interaction occurring in cis and trans in the interior of the interchromatin space (adapted by permission from Macmillan Publishers Ltd: Fraser and Bickmore, 2007, copyright 2007). The model argues that functionally important gene interaction can take place anywhere in this nuclear compartment. In contrast, the CT–IC model predicts that genes are localized within the PR (cf. Figs. 1.4 and 1.2A).

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perichromatin fibrils detach from chromatin and move into the interchromatin space (Puvion and Moyne, 1978), whereas others may move within the PR toward the nuclear pores. In addition to perichromatin fibrils, perichromatin granules are typical RNA-containing nuclear constituents observed within the PR. Neither the formation nor the role of these granules has been clearly demonstrated to date. It has been shown that some perichromatin fibrils are able to form perichromatin granules, which may play a role in the storage/export of messenger RNA (Fakan, 2004; Vazquez-Nin et al., 1997). Several groups analyzed Poly(A)RNA movements in the nucleus of living cells (Dirks and Tanke, 2006; Gorisch et al., 2005; Misteli, 2008; Politz et al., 2006). Shav-Tal et al. (2004) found that nucleoplasmic RNA moves by simple diffusion and in no-directional manner, while Zachar et al. (1993) suggested a channeled diffusion for intranuclear transport of premRNA throughout the Drosophila polytene nucleus (Kramer et al., 1994). Poly(A)RNA moves equally well in speckles and in surrounding nucleoplasm (Politz et al., 2006) and the movement is not disturbed when transcription is blocked (Molenaar et al., 2004). In studies of U2OS cells, Shav-Tal et al. (2004) and Molenaar et al. (2004) found that diffusion coefficients of the predominant RNA fraction in the nucleus ranged from 0.01 to 0.09 mm2/s, whereas Politz et al. (2006) found a coefficient 5- to 10-fold larger in HeLa cells. In addition to this predominant fraction, an immobile or very slowly moving RNA population was reported (Molenaar et al., 2004; Politz et al., 2006; Shav-Tal et al., 2004). The question of whether RNA movements in the nucleus are energy-dependent or not has remained a controversial topic. Molenaar et al. (2004) claimed that RNA mobility requires energy, while Shav-Tal et al. (2004) and Politz et al. (2006) argued that it does not. When following single RNA molecules in living cells, Vargas et al. (2005) proposed that mRNP complexes moved by diffusion through the interchromatin space but needed energy to resume their motion after they became stalled. 3.2.9. Nuclear topography of DNA replication The topography of DNA replication has been studied extensively by methods of ultrastructural cytochemistry combined with EM autoradiography and later with immunocytochemistry. Early EM studies showed that DNA replication does not require an association with the nuclear membrane (Fakan et al., 1972; Huberman et al., 1973; Ockey, 1972). EM studies using short pulses with labeled nucleotides revealed DNA replication sites in the PR regardless of the size of chromatin domains (Fakan and Hancock, 1974; Jaunin and Fakan, 2002). During pulse-chase experiments, newly synthesized DNA was rapidly relocated from the PR into the interior of chromatin clumps, suggesting a permanent movement of DNA between the periphery and interior of chromatin domains in the course of the

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synthetic period ( Jaunin et al., 2000). As in the case of transcription, we tend to assume a stepwise process of chromatin de- and recondensation. Small, decondensed segments of chromatin prone for replication expand into the PR, become replicated, and are subsequently reconfigured as a higher order structure. Although movements of replication machineries along DNA strands cannot be ruled out, it is also possible that replication machineries are fixed within the PR whereas DNA is moving. In HeLa cells (Hozak et al., 1994; Philimonenko et al., 2006), DNA replication was reported to take place in nuclear body-like structures called replication factories. Further studies with improved imaging methods are necessary to refine the dynamic topography of chromatin and essential machineries involved in both transcription and DNA replication, and to explore the structural implications of mechanisms that restore or modify chromatin organization after DNA replication (Corpet and Almouzni, 2009). 3.2.10. Nuclear topography of DNA repair In contrast to the intensely studied biochemistry of DNA repair and factors involved in these processes (Brugmans et al., 2007; Caldecott, 2008; Feuerhahn and Egly, 2008; Hsieh and Yamane, 2008; Lieber, 2008; Peng and Karpen, 2008; Wyman and Kanaar, 2006), the nuclear topography of DNA repair (and possibly also of repair of epigenetic damage) have been explored much less to date (Aten et al., 2004; Falk et al., 2007; Folle, 2008; Krawczyk et al., 2008; Misteli and Soutoglou, 2009; Mortusewicz et al., 2005, 2006, 2008; Solimando et al., 2009). Machineries for the repair of DNA may be built up directly at the nuclear sites where the damage is inflicted. Alternatively, it seems possible that repair machineries are first assembled at other sites and then moved to sites of damage or that damaged DNA/chromatin needs to be repositioned to a nuclear compartment favorable for the execution of repair. Recent evidence has supported the hypothesis that the PR—in addition to its roles in DNA replication and transcription—also serves as the preferential nuclear compartment for DNA repair. An electron microscopic investigation of nucleotide excision repair (NER) in human cell lines has recently shown that following UV-irradiation, XPA and XPC, two proteins involved in the chromatinassociated NER complex, accumulate within the PR. In contrast to XPA, significant amounts of XPC were also found in the compact interior of chromatin domains (Solimando et al., 2009). The authors speculate that both XPA and XPC may be essentially required to execute NER within the PR, but that XPC may play an additional role in the recognition of damaged DNA in the interior of compact chromatin domains. Accordingly, they hypothesize that damaged sites produced in the interior must first be moved to the PR. Notably, the resolution of conventional light microscopy is not sufficient to detect the relocation of damaged DNA (<250 nm) as suggested by Solimando et al. (2009). These new observations emphasize the importance

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to study the topography and kinetics of DNA repair in space and time (4D) at the ultrastructural level. It is not yet known, whether the PR, similarly to its role in excision repair, presents the preferred nuclear compartment for DNA double strand break (DSB) repair. Laser and ion microbeams have provided particularly useful tools to study the topography of DNA repair in single cells. Early laser–UVmicrobeam studies (l ¼ 257 nm) demonstrated the possibility to induce targeted DNA-photolesions yielding excision repair restricted to CTs exposed at a microirradiated nuclear site. As expected, the induction of sister chromatid exchanges and chromosome aberrations was restricted to mitotic chromosomes bearing the damaged DNA (Cremer et al., 1982a,b; Raith et al., 1984; Zorn et al., 1979). Unexpectedly, however, posttreatment of microirradiated cells with caffeine (1–2 mM) synergistically enhanced chromosome damage yielding mitotic cells with ‘‘shattered’’ chromosomes (Zorn et al., 1976). Caffeine has long been known as an inhibitor of postreplication repair and more recently was found to inhibit the kinases ATM and/or ATR (Johansson et al., 2006). In some cases the ‘‘shattering’’ effect appeared restricted to mitotic chromosomes bearing the microirradiated chromatin (partial chromosome shattering, PCS). In other cases, however, the whole chromosome complement appeared ‘‘shattered’’ (generalized chromosome shattering, GCS). Apparently, chromosomal complements with PCS and GCS resulted from a failure of chromosome condensation rather than from extensive DNA fragmentation (Cremer and Cremer, 2006b). In UV-microbeam experiments, where a given incident energy was either concentrated to a small part of the nucleus or distributed over approximately the whole nuclear area, the fraction of mitotic cells with GCS obtained after posttreatment with caffeine increased with the total incident energy and was independent of the distribution of repair sites (Cremer et al., 1981). As an attempt to explain this unexpected phenomenon, a factor depletion model was proposed (Cremer and Cremer, 1986). It argues for a limited pool of certain, still unknown factors, which are recruited at sites of DNA repair, but also involved in the ‘‘maturation’’ of replicated chromatin necessary for a proper condensation process of mitotic chromosomes. GCS occurs under conditions where the available factor pool becomes exhausted. More recently, laser-microbeam experiments were used to study the space–time organization of single and double strand DNA repair (SSBs and DSBs) in nuclei of living cells (Lukas et al., 2005). Prior to microirradiation, DNA was sensitized by the incorporation of halogenated thymidine analogs and in vivo staining with the dye Hoechst #33258. Microirradiation experiments of nuclear areas with focused laser light in the UVA range demonstrated the rapid formation of g-H2AX foci as well as the accumulation of repair proteins, such as Nbs1 and Rad51, at nuclear sites carrying the microirradiated chromatin (Lukas et al., 2003, 2004; Tashiro et al., 2000;

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Walter et al., 2003a). When cells were fixed 30–90 min after microirradiation of nuclei with letter-like patterns, Rad51 accumulations still revealed the form of these letters. This result argues against major movements of microirradiated chromatin during this period. Focused X-rays (Folkard et al., 2001) or focused beams of energetic protons or heavy ions have also been used for the targeted irradiation of nuclei in living cells (Barberet et al., 2005; Folkard et al., 2001; Frankenberg et al., 2008; Greif et al., 2004; Hauptner et al., 2004; Heiss et al., 2006). A series of DNA single and double strand breaks (SSBs and DSBs) can be produced along the route of a given ion through the nucleus providing unique possibilities to study the assembly and disassembly of repair factors at sites of SSBs and DSBs, as well as the recruitment and subsequent release of repair factors involved in DSB repair. At sites of targeted SSBs and DSBs, the rapid formation of g-H2AX was observed due to the phosphorylation of H2AX Ser 29 by the kinase ATM (Hauptner et al., 2004). Indirect immunocytochemistry on cells fixed at different time points after targeted ion-beam microirradiation at different nuclear sites demonstrated the accumulation of factors involved in DSB repair, such as 53BP1, Mdc1, and Rad51, within a few minutes after ion-microbeam radiation of selected nuclear sites. The patterns of accumulated repair factors induced by multiple targeted hits of single ions remained visible for many hours arguing against large-scale chromatin (Hauptner et al., 2006) (Fig. 1.7). A

0.5 h

10 mm

B

15 h

10 mm

Figure 1.7 Microscopic observation of g-H2AX accumulations following ionmicrobeam irradiation. (A, B) Multiple ion-microbeam irradiation with single 100 MeV 16O ions was performed to generate stripes of damaged chromatin in nuclei of living HeLa cells with a distance of about 10 m (Hauptner, 2006; published with permission of the author). Cells were fixed 30 min (A) and 15 h later (B). g-H2AX generated by ATM-catalyzed phosphorylation of serine 139 of the histone variant H2AX was detected by indirect immunofluorescence with anti-g-H2AX-specific antibodies. Notably, g-H2AX positive stripes could still be detected 15 h after ion-microbeam irradiation with a similar geometry as in cells fixed after 30 min. Notably, the stripes detected after 30 min were still mostly parallel in different nuclei. In contrast, the stripes detected after 15 h revealed different directions in different nuclei indicating a rotation of nuclei (Hauptner et al., 2006).

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The sequential generation of DSBs at selected nuclear sites with chosen time intervals was employed to study at the single cell level, whether earlier and later generated DSBs may compete for repair factors (Greubel et al., 2008a,b). Competition may be brought about by different on/off kinetics affecting the binding and release of different repair factors at DSBs, different pool sizes, synthesis and degradation of repair factors as well as a dynamic sequence of chromatin modifications at sites of DSBs. Tight binding of certain repair factors for prolonged times at sites of DSB repair generated earlier in a nucleus may result in the undersupply of such factors at additional sites of DSBs generated later. The experiments described above suggest such a competition effect between earlier generated and later generated DSBs for the repair factors 53BP1 and Rad51. Most recently, transgenic human cell lines (HeLa, U2OS) were produced, which express one of the GFP-tagged DSB repair proteins Rad5, Rad52, 53BP1, and hMdc1. These cell lines were used to follow directly the recruitment of repair proteins to ion-microbeam irradiated nuclear sites with DSBs in the nuclei of single living cells (Hable et al., 2008). Visible recruitment of Mdc1-GFP to sites of DSB repair was already detectable 10–20 s following ion-microbeam irradiation. Mdc1 directly binds g-H2AX (Stucki et al., 2005). In contrast, visible recruitment of Rad51GFP and Rad52-GFP required 10–15 min. These preliminary results underline the potential of ion-microbeam studies to investigate the sequence and kinetics of the recruitment of repair factors.

4. Perspectives: Open Questions and Possibilities to Answer Them 4.1. Studies of higher order chromatin arrangements Despite the evidence spelled out above for some evolutionary widely conserved features of nonrandom higher order chromatin arrangements, it is important to note the astounding variability of such arrangements both with regard to different cell types of a given organism (Hoffmann et al., 2007; Olins et al., 2008; Solovei et al., 2009) and species-specific peculiarities. In this context it is important to study nuclei in a wide range of eukaryotic species from distant parts of the evolutionary tree, including nuclei with strikingly different phenotypes, such as macronuclei of Ciliate species (Olins et al., 1981; Postberg et al., 2005, 2008), nuclei of dinoflagellates (Moreno Diaz de la Espina et al., 2005), nuclei in endocycling cells of the urochordate Oikopleura dioica (Costas and Goyanes, 2005; Spada et al., 2007), and nuclei of Trypanosoma brucei (Navarro et al., 2007). It is important to be aware that evidence for a nonrandom spatial proximity of CTs (or chromosomal subregions down to individual genes)

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does not prove that this proximity is required for a specific functional interaction. Deviations from a random arrangement may have simple and functionally irrelevant causes. For example, a different arrangement of large and small CTs may be brought about by different geometrical constraints acting in nuclei with different shapes (Bolzer et al., 2005). At this point it is not known whether mechanisms responsible for different nuclear shapes act from inside the nucleus and/or from outside. For example, the shape and compaction of CTs may affect the shape of the nucleus. Alternatively, the nuclear shape may be modified by interactions of the nuclear envelope with the cytoskeleton and even an extracellular matrix (Gieni and Hendzel, 2008). Understanding the functional implications of nuclear architecture (s) and mechanisms necessary to achieve, maintain, and alter higher order chromatin arrangements during development and cell differentiation is still a far-off goal. Present knowledge about higher order chromatin structures and their arrangements in different cell types of a given organism and similarities, as well as differences of such arrangements in corresponding cell types of different species are still too fragmentary to distinguish evolutionarily shared principles, which may be valid in all eukaryotes, from cell type- and species-specific differences (Foster and Bridger, 2005).

4.2. Nuclear topography and models of the nuclear architecture We need to integrate the level of nuclear architecture with the levels of epigenetics and of genome organization (Fig. 1.1) (Varga-Weisz and Becker, 2006; Wallace and Felsenfeld, 2007) and define the components essential for higher order chromatin organization and its changes due to internal and external stimuli (Cai et al., 2006; Carter et al., 2002; Galande et al., 2007). Possible implications of nuclear architecture for the ability of proteins to locate specific target sequences or structures among a vast excess of nonspecific DNA are not understood (Cremer et al., 1993; Gorman and Greene, 2008; Kampmann, 2005). We are still ignorant about the possible functional role(s) of an evolutionarily conserved radial distribution of genedense and gene-poor chromatin and cell type-specific proximity patterns, nor do we know the reasons for changes of chromatin arrangements in nuclei of cycling cells and during postmitotic terminal cell differentiation. We do not know whether changes of higher order chromatin arrangements are the cause or effect of functional changes or are even functionally meaningless, not to speak of the mechanism(s) which are necessary to accomplish such changes. One concept, which has obtained prominence during recent years, suggests expression hubs, where the localized position of multiple coregulated genes would facilitate their intercommunication by helping to form and then utilize localized concentration of regulatory

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proteins (Kosak and Groudine, 2004). However, evidence for the structural nature of such hubs is lacking. The different models of nuclear architecture discussed above should not necessarily be considered as mutually exclusive. They may rather accentuate different possibilities, which may be realized in some cell types but not in others. While the presence of CTs in nuclei of multicellular organisms has become a generally accepted feature of models of the nuclear architecture, the concept of a structurally and functionally distinct IC has remained controversial. This concept has been proposed as an essential part of the CT–IC model (Cremer and Cremer, 2001), but disputed by competing models such as the ICN and GLF models. Contrary to other models we argue that the IC and the PR are two structurally distinct and functionally interacting nuclear subcompartments, and that the PR serves as the nuclear subcompartment for transcription and RNA splicing, as well as DNA replication and according to most recent evidence also for repair (see above). Figure 1.8 illustrates the contrasting views of the functional nuclear architecture proposed by the CT–IC model (Fig. 1.8A) as compared to a chromatin loop model arguing for numerous large chromatin loops with a thickness of 11 and 30 nm, respectively (Fig. 1.8E). According to the CT–IC model one would expect that snapshots of the chromatin organization of nuclei at any given time point should only reveal a relatively small fraction of chromatin present as extended 11 and 30 nm chromatin fibers or loops, whereas the ICN and GLF models argue for an extensive contribution. The space–time structure of 1 Mb domains or clusters thereof (Fig. 1.8A) as well that of higher order chromatin fibers is unknown. The CT–IC model speculates that (a) each 1 Mb domain is built up from a series of  100 kb chromatin loop domains and (b) the smallest branches of the IC end within 1 Mb chromatin domains (Cremer and Cremer, 2006b), but compelling experimental evidence is lacking. In particular, we do not know how much DNA is present in linearly arranged chromatin fibers of different compaction levels. With diameters of only a few hundred nanometers, the structure of 1 Mb chromatin domains cannot be imaged by experiments using the resolution of conventional light microscopy. The CT–IC model takes into account perichromatin fibrils as in situ forms of single nascent hnRNA transcripts being produced in the PR. Aggregates of PFs may reflect the ongoing, independent transcription of several closely adjacent genes or multiple transcripts on the same gene. Such multiple transcripts were observed with EM on spread transcription complexes from different cell types (Foe et al., 1976; Harper and Puvion-Dutilleul, 1979; McKnight and Miller, 1979; Puvion-Dutilleul et al., 1978). The chromatin loop model takes into account TFs, which can transcribe several genes simultaneously. TFs are located within fields of intermingling chromatin loops, protruding from condensed chromatin domains. These different views of the nuclear topography of transcription cannot easily be

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A Interchromatin Chromatin domains compartment

E Nuclear envelope

Speckle

B

F IC

C

G

D

H

Figure 1.8 Schematic drawing comparing two models of transcription organization within the in situ nuclear landscape. (A) A scheme of a partial nuclear section according to the CT–IC model (cf. Fig. 1.2A) with 1 Mb chromatin domains and clusters of several such domains, as well as the interchromatin compartment expanding between them with splicing speckles and nuclear bodies. An enlargement of the boxed insert is shown in (B) and (C). (B–D) According to the CT–IC model, major transcriptional activity is expressed in the PR occurring on the border of chromatin domains (roughly delimited by the red dotted line). In the case where the chromatin domains (in gray) are close to each other (within a distance of about 400 nm or less), Brownian chromatin movement may shortly put transcription sites together (B and D, blue dotted line) and then separate them again (C). (E) This partial nuclear section differs from the situation presented in (A) by the assumption that the interchromatin compartment in addition to splicing speckles and nuclear bodies contains numerous chromatin loops expanding from higher order chromatin domains, as well as transcription factories. An enlargement of the boxed insert is shown in (F) and (G). Note that a perichromatin region (PR)

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reconciled. Evidence that RNA Pol II is enriched in the PR (Cmarko et al., 1999) raises the question whether large TFs, seen after immunostaining of RNA Pol II (Sexton et al., 2007), could be reconciled with the perichromatin compartment model of transcription by the assumption that they represent a chromatin domain with RNA Pol II labeled in the PR surrounding the compact core of this domain; the fact that the whole domain shows fluorescent staining could simply be caused by the limited axial resolution. Smaller TFs may consist of aggregates of perichromatin fibrils. If so, we would expect that such TFs should be localized in the PR rather than in the middle of a field of intermingling chromatin loops. The TF hypothesis argues for a single structural unit, which serves several genes simultaneously. In the case of the chromatin loop model, genes being transcribed within a single TF will stay together despite Brownian chromatin motions (cf. Fig. 1.8F with G). It was reported that TFs visualized as RNA Pol II foci could be detected as persistent structures even in the absence of transcription (Mitchell and Fraser, 2008). In our view, a cluster of perichromatin fibrils may represent a transient spatial association without a functional necessity to keep this association for a prolonged time. Accordingly, Brownian motion of chromatin domain can quickly dissociate an aggregate of PF mimicking a TF (cf. Fig. 1.8B with C). An elegant ultrastructural study shows that global downregulation of transcription quickly leads to a strong reduction of the number of PF; a restimulation of cellular RNA synthesis gives rise to a rapid occurrence of PF which parallels that of the newly formed nucleoplasmic RNA within the PR (Nash et al., 1975). We do not know the true space–time chromatin structure of either a silent or a transcribed gene in vivo, not to speak about the implications of transcription on neighboring chromatin. Some speculations seem justified at

does not exist according to this scheme. Even the demarcation between higher order chromatin domains and the interchromatin compartment (cf. Fig. 1.3; Rouquette et al., 2009) may become abolished in case of more extensive outlooping of chromatin. Comparative studies of chromatin topographies with ultrastructural resolution are necessary in nuclei from different species and cell types with high and low global transcription rates in order to test whether transitions states between the nuclear topographies shown in (A) and (E) exist. (F–H) Transcription factories (TFs) (blue) are located in the interchromatin space expanding between chromatin clusters (cf. Fig. 1.6A). Several large chromatin loops containing active genes pass through TFs (cf. Fig. 1.6A). Locally constrained movements of chromatin clusters do not basically change the topography of the loops simultaneously recruited by a given TF. IC: interchromatin compartment; small blue dots: RNA polymerase II; gray loops: DNA containing transcribed sequences emerging from chromatin domains; short green filaments: perichromatin fibrils (RNA transcripts) loaded with hnRNP proteins illustrated in red; black dotted line circles indicate places of partial chromatin decondensation taking place in the PR. Bars: (A, E) 500 nm; (B, C, F, G) 200 nm; (D, H) 60 nm.

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this point, if only to trigger thoughts and new experimental approaches concerning the still unknown extent of local chromatin dynamics possibly involved in transcription (as well as in DNA replication and repair). We will consider two scenarios for the scale and dynamics of chromatin reorganization and repositioning events possibly involved in transcription. Although these scenarios are admittedly highly speculative, we wish to undertake this exercise in order to show how little we still know about the structural biology and nuclear topography of transcription as one of the most important nuclear processes. 

Scenario 1. Full decondensation of genes as an essential requirement for transcription.

A whole gene present in a higher order chromatin configuration in its silent state may decondense, prior to transcription, to a looped  30 and 11 nm chromatin fiber expanding within the PR or into the interior of the IC (depending on one’s favored model of nuclear architecture, Fig. 1.2). This assumption would fit models, which argue for the presence of transcriptionally active genes on extended chromatin loops, but may turn out to be oversimplified if not invalid to describe the true complexity of gene regulation. Whereas the ICN model (Fig. 1.2B) seems to argue that a transcription machinery is built up wherever necessary, we argue that its built up and spatially fixed predominantly within the PR (Fig. 1.2A). Active genes may be entirely located within the PR possibly expanding along the surface of the compact chromatin domain interior, while silent genes are buried entirely in the interior including its regulatory sequences. Such an organization may provide a topographical barrier, which helps to prevent a functionally detrimental or even catastrophic event of transcriptional activation of permanently silent genes, since transcriptional activation of such genes would require their repositioning into the PR. In order to work, such a mechanism would require that such a buried gene is not accessible for the transcription machinery. Since published data indicate that the interior of chromatin domains is accessible for individual proteins, it seems plausible that a factor required for the relocation of a buried gene into the PR can reach its respective target within the interior of a given domain. Alternatively, we consider the case that a regulatory sequence responsible for the positional transfer of the buried part of the gene may always be located in the PR, like the bells at house walls (chromatin domain surfaces) located along a given street (interchromatin channel), whereas intron and exon sequences are buried in the domain interior. 

Scenario 2. Transcription involves stepwise gene decondensation and recondensation process.

In case that all silent genes independent of the location of the major chromatin part expose a target site within the PR, this site could become the starting point for the sequential, stepwise relocation of only a small, fully

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decondensed segment of a given gene into the PR during transcription, while the nondecondensed part is still located either directly beneath the PR or even deeper within the compact interior of a chromatin domain. The accessibility of the domain interior for individual proteins involved in the construction of a complete transcription competent machinery is consistent with the possibility that such a machinery can be built up exclusively in the PR since only decondensed chromatin in the PR may show chromatin modifications required for the binding of certain transcription factors essential for its builtup. Scenario 2 argues for a transcription process working hand in hand with a very dynamic de- and recondensation process of a gene’s chromatin in a sequential, stepwise manner. While one piece of DNA is being transcribed, the next segment of the gene is becoming fully decondensed and located into the PR, while the posttranscriptional piece of DNA becomes recondensed and relocated into core chromatin beneath the PR. Accordingly, transcription would involve highly dynamic local chromatin events albeit at scales which cannot be resolved by conventional light microscopy. For both scenarios a nonrandom organization of the chromatin domain is an essential requirement raising the question of which mechanism may guide the movement of a buried gene or part of it to the domain surface and back into its interior? Finally, it should be emphasized that we do not even know presently whether the PR is the preferential nuclear subcompartment for active genes only or whether it contains both active and silent genes. Observations suggesting the presence of silent PcG genes within the PR provide a case in point (Cmarko et al., 2003). Whatever scenario may be considered more likely than others, we need to know to what extent certain sequences are nonrandomly exposed at chromatin domain surfaces, while others are located in the interior. We also need to know the circumstances and dynamics with which certain genes or parts thereof may be exposed in the PR and relocated in the interior of chromatin domains. Timescales required for these dynamics may differ strongly between genes which become transcriptionally active on short notice and genes which are kept permanently silent in a given somatic cell type, such as pluripotency genes which become only active during a complex reprogramming event. Since the scale of functionally essential repositioning events may often range below the resolution of conventional light microscopes, attempts to obtain experimental evidence in favor of or against the transcription scenario discussed above can only be based on EM possibly supported by advanced light microscopic approaches breaking the Abbe/Raleigh limit of conventional light microscopy. Studies of the dynamic nature of nuclear architecture are still in their infancy and many pertinent questions remain to be answered. When are cell type-specific proximity patterns of chromosomes established during development (during mitosis, interphase, postmitotic terminal differentiation)? Can a given proximity pattern be maintained throughout mitosis?

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Or is such a pattern lost during mitosis and needs to be newly established during interphase or in postmitotic cells? What kind of mechanism allows different cell types to adopt cell type-specific chromosome proximity patterns? Obviously, direct observations of CT and gene movements in living cells are desirable. Such experiments are possible, but are not easy to perform, since the introduction of visible markers may interfere with normal gene movements. For a comprehensive answer, new, sophisticated approaches which combine 4D (space and time) live-cell studies of nuclei with ultrastructural resolution must be developed.

4.3. Forces responsible for changes of higher order chromatin arrangements What are the forces which drive short-range (nm scale) and long-range (m scale) movements of nuclear structures? For short-range movements, Brownian motions may entirely suffice, although even short-range movements may be energy-dependent. The problem of energy dependence is particularly obvious in case of long-range movements of chromatin, which bring together DNA from widely distant parts of a given chromosome or even from different chromosomes. Evidence for the nuclear localization of actin and myosin has put these proteins in the spotlight as potential participants in long-range chromatin movements (Farrants, 2008; Milankov and De Boni, 1993) and their important roles in transcription and possibly in signal transduction were revealed (Hofmann, 2009; Louvet and Percipalle, 2009; Pederson, 2008; Pestic-Dragovich et al., 2000). Nuclear actin/myosin as a nuclear motor machinery has recently been claimed as being involved in the molecular mechanism(s) responsible for large-scale movements of CTs, chromatin domains and genes (Hu et al., 2008; Mehta et al., 2008). Yet, we are still far away from a true understanding what mechanisms actually drive these movements. In order to achieve an impression of the potential complexity of a mechanism required for ‘‘kissing’’ events in trans, let us consider two scenarios (A and B). Scenario A: CTs harboring genes, which engage in trans ‘‘kissing’’ events in a given cell type, maintain a strictly nonrandom neighborhood directly favorable for such a ‘‘kiss.’’ Under such conditions, small-scale Brownian motions may suffice to bring sequences into ‘‘kissing’’ contact (here even positional changes as small as 50 or 100 nm may be of great functional significance). When needed, random contacts can be stabilized by protein– DNA interactions. Scenario A does not conform to present data suggesting a strong cell-to-cell variability of CT proximity patterns, but given the relative paucity of pertinent studies it should not be excluded from consideration. In favor of this scenario we need to answer the question as to which mechanisms are responsible to establish cell types with a highly ordered chromatin neighborhood?

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Scenario B: Potentially ‘‘kissing’’ sequences are widely separated from each other (typically in the order of several m). Following mitosis the CT neighborhood arrangements present in daughter nuclei differ largely and unpredictably from the arrangement present in the mother nucleus. Accordingly, two CTs, which were neighbors in the mother nucleus, may be widely separated in its daughters. Since both 3C and 3D FISH experiments provide only snapshots from a population of cell types, transient ‘‘kissing’’ events can be detected only in a minority of nuclei. Longdistance movements of either whole CTs or of giant loops expanding from them (Fig. 1.2D) are necessary to bridge the space between remote CTs. Mechanism(s) necessary to bring about cell type-specific, transient or permanent ‘‘kissing’’ events in trans over long distances must involve major—and as we argue: directed—chromatin movements (Chuang et al., 2006). Recent claims that entire CTs are able to make major movements within periods of half an hour or even less (Hu et al., 2008; Mehta et al., 2008) came unexpected and were so far not supported by direct observations of such rapid movements in living cells. In case that several ‘‘kissing’’ events between different pairs of homologous or nonhomologous CTs are required, the required mechanism(s) may turn out to be exceedingly complex. To illustrate this point, let us assume that CT neighborhood arrangements are random in a given stem cell population, whereas different somatic cell types attain cell type-specific proximity patterns of chromatin to allow DNA sequence interactions in trans. Trans ‘‘kissing’’ events may be brought about by large-scale movements of entire CTs and/or of giant chromatin loops expanding from the respective CTs. In case that a congression of entire, widely separated CTs is required in order to enable a ‘‘kiss’’ in trans, repositioning would necessarily involve numerous other CTs as well, since CTs, which separate the two potential ‘‘kissers,’’ need to move aside. In case of long-range interactions of giant chromatin loops (Fig. 1.2D) (de Laat, 2007; Ling and Hoffman, 2007), physical constraints need to be overcome that will hinder long-distance passages of giant loops through the nuclear space. On their route, giant loops must be able to penetrate through or pass around one or several other CTs forming obstacles between the site of departure and the site of arrival of a gene traveling on a given giant loop. In principle, interchromatin channels could serve as routes for expanding loops to remote nuclear sites (Albiez et al., 2006; Bouchet-Marquis et al., 2006). However, such channels are, of course, not empty but filled with macromolecules and nonchromatin domains, which also provide obstacles for expanding and retracting giant chromatin loops. Models contradicting the existence of the IC, such as the ICN and the GLF model, make the directed passage of giant loops throughout the nuclear space an even more insecure and doubtful journey. In cycling cells, chromosome condensation at the onset of mitosis may create additional mechanical problems for the undisturbed retraction of

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such loops toward their home chromosome. We conclude at this point that the topographical implications of transient ‘‘kissing’’ events have not been sufficiently described to date nor has compelling evidence been provided for a direct functional significance of such events.

4.4. Unexplored biophysical properties of nuclei The biophysical properties of nuclei are another field, which needs to be explored (Kanger et al., 2008; Lavelle and Benecke, 2006; Rowat et al., 2008). Changes of nuclear shape and stiffness during differentiation from stem cells to terminally differentiated cells are well known but not well understood (Pajerowski et al., 2007). Evidence that CTs apparently form 3D higher order chromatin networks (Albiez et al., 2006) may have important, still unexplored implications for nuclear stiffness and mechanotransduction. The latter implies that forces transmitted from the extracellular matrix via the cytoskeleton to the nucleus can alter gene expression in a variety of cell types (Campbell et al., 2008; Gieni and Hendzel, 2008). The nucleus provides a crowded environment. The role of macromolecular crowding and entropic forces on local chromatin compaction and thus functional states of chromatin is supported by both theoretical considerations and experimental evidence (Hancock, 2004; Hancock, 2007; Richter et al., 2008; Rippe, 2007). This evidence suggests that macromolecular crowding is involved in self-association of polynucleosome chains (Hancock, 2008) and stabilizing CT structure. It seems possible that CTs represent separate phases like those seen in heterogeneous particle mixtures by experiment and simulation. Yet, the interplay of these factors is not well understood with respect to the compaction or decondensation of different chromatin domains containing unique and/or repetitive DNA sequences, nucleosomes with different epigenetic modifications, and different histone variants as well as countless nonhistone proteins. It is essential to combine microscopy with biochemical and biophysical approaches to achieve a better understanding how different nuclear domains are formed, maintained, altered, or degraded in the context of the global functional architecture of the cell nucleus.

4.5. Summary Although often labeled as ‘‘only’’ descriptive, detailed structural studies are mandatory to provide secure knowledge of evolutionary-conserved main structural motifs and of the extent of cell type- and species-specific features. Such data are indispensable as an essential basis of future research, even if the results cannot be used immediately to generate or test molecular hypotheses on how a given cell type-specific nuclear architecture is generated and maintained. Like extensive comparative DNA sequencing data are

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necessary to understand the history of species evolution, comparative evolutionary data of nuclear architecture are an indispensable part of attempts to understand how the present range of epigenomes of cell types within and between species has evolved. The planning of such studies on a sufficiently large scale requires improved bioinformatics tools able to handle the analysis of large amounts of data. In comparison with projects, which focus on the evaluation of molecular mechanisms, chances of getting such ‘‘descriptive’’ projects funded, as well as chances to publish their results in high impact journals, are presently much lower. This situation provides an important drawback for the whole field of cell biology. Without detailed descriptive knowledge on the possible extent of modifications of the nuclear architecture, speculations about mechanisms involved in generating, maintaining, or even reprogramming the myriad of epigenomes present in multicelluar organisms are built on insecure ground. Current experimental approaches to manipulate the nuclear architecture (Finlan et al., 2008; Kumaran and Spector, 2008; Mateos-Langerak et al., 2007) need to be further developed in order to manipulate the location and/or compaction of entire CTs, chromosomal subregions, or single genes with the aim of exploring possible effects of such changes on nuclear functions. These approaches should be pursued together with more sophisticated, quantitative modeling of higher order chromatin and nuclear architecture at large, which takes into account the dynamic interactions of nuclear components including the higher order organization of chromatin and chromatin interactions, the movements of important molecular constituents, such as RNA molecules and proteins with regulatory functions, as well as the assembly and disassembly of functional machineries, nuclear bodies, and splicing speckles (Carrero et al., 2006; SaccoBubulya and Spector, 2002). Only few attempts have been made so far to develop models of individual CTs and their higher order organization, which allow quantitative predictions for experimental tests (Bolzer et al., 2005; Cremer et al., 2000; Kreth et al., 2004b; Munkel and Langowski, 1998; Munkel et al., 1999; Sachs et al., 1995; Shopland et al., 2006), such as quantitative predictions on experimentally induced chromosome aberrations. Such experiments in turn will help to falsify or validate and improve such models (Branco and Pombo, 2006; Friedland et al., 2008; Holley et al., 2002; Kreth et al., 2004a, 2007; Levy et al., 2004). As important as a valid theoretical foundation for a research strategy are questions concerning the usefulness and limitations of available experimental methods and the development of new methods, which open the gate for new discoveries. In Section 5 we will focus on new microscopic developments and the importance of being earnest about correlative microscopy combining both advanced FM and EM. Concluding this section, we wish to point out that it is not helpful to emphasize one approach on the cost of another. Understanding the

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functional nuclear architecture is a huge task for many years to come. While many tools have still to be developed, integrative approaches are the key to success. It is obvious that approaches dominated by a single method lead to insufficient answers or fail entirely (Robinson et al., 2007). The importance of multidisciplinary approaches can be underlined by progress in understanding the structure–functional relationships of nucleoli, probably still the most studied nuclear compartment/entity to date (McKeown and Shaw, 2009), the nucleosome (Corpet and Almouzni, 2009; Luger et al., 1997), and the NPC (Alber et al., 2007b; Beck et al., 2007; Dange et al., 2008; Maco et al., 2006). This progress has become possible by the integration of structural analyses with a range of complementary methods as well as data translation into models for spatial restraints, optimization, and ensemble analysis (Alber et al., 2007a). Despite this progress there are still many open questions and conflicting ideas how these so far best studied nuclear structures really function. Following these examples, functional and structural data from cells and their nuclei must be obtained by all possible means from the level of single molecules to a multitude of higher order chromatin structures to nonchromatin domains, such as nuclear bodies and interchromatin granules (splicing speckles) and last but not least to the macromolecular machineries involved transcription, RNA-processing, replication, and repair. The main strength of a proper combination of state-of-the-art microscopic methods lies in its potential to elucidate the dynamic topography of all nuclear constituents at the single cell level, but this can only be exploited when imaging methods are part of a proper context of other methods.

5. Quantitative Microscopic Analysis of Nuclear Architecture This section was written in order to provide basic information about special advantages and limitations of currently available microscopic approaches and to point out some new developments. While this information is, of course, limited, we hope that it provides an idea of the complexities of advanced cell imaging techniques and their potential for future studies of the functional nuclear architecture.

5.1. Electron microscopy Compared with light microscopy, the most obvious advantage of EM is its much higher resolution, which is in the range of 0.5 nm in the case of TEM and a few nanometers in the case of scanning EM, while the most obvious disadvantage is the restriction to fixed cells only. Methods for the

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visualization of DNA or RNA synthetic sites use either tritium labeling and autoradiography or halogenated precursors and immuno-EM. In both cases the specimen is represented by ultrathin sections of a chemically fixed or cryofixed cellular sample either subsequently embedded into plastic resin or cryosectioned. In the former, visualized label reflects the intracellular distribution of nucleic acids localized by virtue of a radiation-sensitive photographic emulsion superposing the specimen and photographically treated to obtain the final signal. Although offering a lower detection resolution than immuno-EM, it is especially convenient for subsequent kinetic analyses of distribution of different components in the cell. As to the immunocytochemical detection, it is important to be aware of the difference of structural preservation between pre- and postembedding labeling approaches: 1. In a preembedding protocol a given cell is embedded in resin for subsequent ultramicrotomy only after completion of immunocytochemical reactions with antibodies against specific epitopes and, for example, a secondary antibody coupled with a convenient marker, typically giving rise to a product of an enzyme–substrate reaction. Accordingly, ultrathin sections studied by EM reveal specific signal (and unwanted background) throughout the whole section. The major disadvantage of preembedding protocols stems from the fact that procedures needed for performing immunocytochemical labeling before embedding can damage the ultrastructure of the cell to an extent which is difficult to control precisely. Permeabilization steps with detergents are often required to obtain probe penetration into the sample, which may give rise to extraction or displacement of cellular components. Structural damage due to DNA dispersal is particularly obvious in the case of in situ DNA hybridization procedures, which require a DNA denaturation step (Solovei et al., 2002). In all cases where it is necessary to generate cytochemical signal from the entire depth of each section, the preembedding approach cannot be avoided, but it is necessary to emphasize here the danger, already discussed above, of structural artifacts generated by preembedding in contrast to postembedding protocols. 2. In a postembedding protocol the fixed cell is first embedded in resin, and ultrathin sections are prepared before an immunocytochemical method is applied. In addition, chemically fixed and cryosectioned material may also represent a suitable alternative (Fakan et al., 1984). The major advantage of this approach is due to the unparalleled preservation of fixed specimens. Epitopes for binding of specific antibodies or nucleic acid sequences for in situ hybridization with specific DNA or RNA probes are detected only at section surfaces exposed by ultramicrotomy. Structures embedded within the section are not affected by labeling and detection procedures and thus remain well preserved. Compared with

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preembedding protocols, this generally gives rise to lower signals. However, preembedding labeling is achieved at the cost of a much lesser and sometimes unacceptable quality of ultrastructural preservation and, consequently, of localization precision. Moreover, reactive sites located inside dense compact cellular compartments may not be accessible for probes, while they are uncovered by sectioning and consequently accessible when the reaction takes place directly on ultrathin sections. For the analysis of finest details of structures revealed by means of ultrastructural cytochemistry, the postembedding approach is recommended. Ultrastructural cytochemical studies of the cell nucleus (Biggiogera and Fakan, 2008) usually require chemical fixation with aldehydes followed either by dehydration and embedding into different kinds of resin, or by cryoultramicrotomy making use of a cryoprotectant (Tokuyasu, 1973). Protocols using cryofixation, cryosubstitution, and resin embedding excluding any conventional fixative can also routinely be applied (von Schack and Fakan, 1993), allowing one to work under optimal conditions with regards to the detection of various molecular components of the cell and its activities. In this context, it is interesting to mention that all these different methods, including cryoelectron microscopy of vitrified sections (Bouchet-Marquis et al., 2006), have yielded similar basic morphological features regarding the compartmentalization of the cell nucleus.

5.2. Far-field fluorescence microscopy with conventional resolution Compared with EM the most obvious advantage of FM is the possibility of live-cell observations, while its much lower resolution represents the most obvious disadvantage. Conventional epifluorescence microscopy (EFM) and confocal laser scanning microscopy (CLSM) are limited to an optical resolution of about 200 nm laterally and 600 nm axially. Vital fluorescence markers or tags have offered the possibility to follow individual labeled cellular components and to analyze their interactions in intracellular molecular pathways in living cells down to the level of single molecules (Grunwald et al., 2008). The caveat and limitation of these studies is the requirement that fluorescence tags must not alter the cellular functions under scrutiny. Structural deterioration must be taken into account in DNA in situ hybridization experiments. Heat denaturation of DNA is typically used as a means to render target DNA strands accessible to hybridization with single-stranded DNA probes. TEM of cells after heat denaturation revealed pronounced, although locally restricted dispersal of DNA. As a consequence, 3D FISH combined with new fluorescent microscopic approaches breaking the Abbe limit (see below) or with subsequent TEM analysis (Solovei et al., 2002) is a priori problematic as an approach to study the

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ultrastructure of chromatin. On the other hand, the relative 3D positions of CTs and 1 Mb chromatin domains recorded in living cells after pulselabeling with fluorochrome-conjugated nucleotides during S-phase hardly changed after 3D fixation procedures with buffered formaldehyde (Solovei et al., 2002). When these fixed cells were subjected to a chromosome paint experiment, changes of the relative positions of foci were noted in the order of a few hundred nanometers. Accordingly, we argue that 3D FISH is a useful approach to detect differences of the architecture of individual CTs, as well as differences of CT arrangements down to the limits of conventional light microscopic resolution. CLSM has provided a tool for the routine recording of stacks of light optical serial sections, which are perfectly suitable for 3D reconstructions. The application of deconvolution algorithms seems mandatory for light optical sections obtained by EFM, but can also improve the quality of CLSM sections considerably. The rational choice, which of various deconvolution procedures should be preferred for a given case, is often hampered by a typical lack of knowledge about what the true features of imaged structures may be. For example, an observer may prefer structures with clear borders distinguishing these structures from their neighborhood. The choice of a deconvolution protocol, which enhances such borders, however, may be misleading. The more already is known by other studies about the true features of a structure, the easier it will be to make a reasonable choice. Multicolor 3D FISH has allowed to visualize multiple targets with different colors, such as the CTs from all pairs of homologous autosomes and the two sex chromosomes X and Y in human diploid fibroblast nuclei (Bolzer et al., 2005). A major disadvantage of CLSM is its limited recording speed, which does not provide the temporal resolution necessary to monitor very rapid biological processes in living cells, for example, the dynamics of microtubule assembly and disassembly. This disadvantage has been overcome by the development of spinning disk confocal microscopy. These instruments contain a thin wafer with hundreds of pinholes arranged in a spiral pattern, called a spinning disk and in contrast to a conventional CLSM where an image is recorded point by point, spinning disk confocal microscope allows the simultaneous recording of fluorescence emitted by many object points. This allows a much higher speed for the recording of a very thin optical section. AOTFs (acoustooptical tunable filters) offer a microseconds switch of excitation wavelengths. It is often important to decide whether two fluorescent domains are fully separated from each other, whether they just touch each other or whether they—within the resolution limits of the microscopic equipment—partially or even fully colocalize with each other. We start with the apparently most trivial request to obtain a random sample for statistical analysis. In case of 3D FISH experiments, some 20–50 cells are typically chosen from the whole

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cell population or from a given cell type present in this population (or in case of more specific demands from cell types at a given stage of interphase or postmitotic differentiation). As a major danger, one should be aware of an unnoticed biased selection of cells in quantitative 3D FISH assays, which may be an important source of misleading conclusions. Only 3D FISH preparations with a very high yield of analyzable cells (> 95%) should be evaluated. Criteria for rejection of cells from further evaluation must be carefully outlined in advance and the reasons for rejection during evaluation should be presented. For obvious reasons, one should strictly avoid looking on FISH signal patterns when deciding which cells are chosen for detailed evaluation. This can be achieved by looking first on the DNA counterstain and rejecting cells with apparently damaged nuclei. Only thereafter filter sets should be changed to record the FISH signals. The use of a random number generator to choose coordinates for evaluation on a given slide, likely provides the best possible assurance against sampling errors, which may lead to wrong statistical conclusions. Secondly, one should keep in mind the limitations of the resolution of the microscopic equipment used for analysis. A conventional confocal microscope, for example, has an optical resolution of about 200 nm in x/y- and 600 nm in z-direction (Stelzer, 1995). Small domains apparently overlapping in a light optical section may be separated by a corridor of another nuclear compartment when viewed with the much higher resolution of the electron microscope. According to the Abbe limit of light optical resolution, two fluorochromes closer to each other than the resolution limit of about half the wavelength used for imaging (Abbe 1873; Rayleigh 1903) cannot be discriminated as separate objects as long as they emit light of the same spectrum. An important implicit assumption of this statement is that the two objects emit light simultaneously. We will see below that this limitation can be overcome in case that the two objects emit light with different spectral signatures, e.g. in a sequential manner (Spectral Precision Distance Microscopy, SPDM; see below). The choice of fluorochromes, which emit fluorescent light of different wavelengths, provides another possibility to overcome this limitation. Here, the emission of the two fluorochromes can be recorded separately by use of an appropriate filter, and the 3D position of the intensity gravity center of the fluorescence can be recorded separately for each fluorochrome with a precision in the nanometer range. This allows high precision 3D distance measurements between the two intensity gravity centers with a resolution clearly far below the Abbe limit (Bornfleth et al., 1998; Cremer et al., 1999). As a caveat of such measurements, one has to take into account that chromatic aberrations of the microscope objective, which may be quite different for different wavelengths, lead to chromatic shifts, which need to be very carefully corrected, but this can be done with high precision (Esa et al., 2000; Rauch et al., 2008). When all the mistakes lurking around quantitative 3D light

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microscopy are carefully avoided, conventional FM in the SPDM mode allows high precision 3D distance measurements as small as 50 nm. In contrast to this improvement of 3D distance measurements between a small number of closely adjacent objects, quantitative measurements of the true shape and volume of objects far below the Abbe limit of resolution is still impossible with conventional FM, including CLSM and one cannot decide to which extent the periphery of two objects may overlap. It is even difficult to perform precise measurements of volumes of fluorescent objects with dimensions clearly above the Abbe limit, such as painted CTs, since these measurements are threshold-dependent. When using paint probes for different CTs, it is important to be sure that they are of comparable quality in covering their target CT. Furthermore, the use of an excess of unlabeled Cot1-DNA to prevent the unwanted hybridization of labeled repetitive probe sequences, means that only a fraction of the entire CT in question can be targeted even with the best chromosome paint probes. The determination of an appropriate threshold able to distinguish specific fluorescence that belongs to a given object, for example, a splicing speckle, from background fluorescence is a difficult problem. Attempts to solve this problem are typically based on segmentation procedures using a threshold, which apparently distinguishes between pixels/voxels with signals above the threshold arguably belonging to the fluorescently stained object, while pixels/voxels below the threshold are attributed to background. Depending on the choice of the threshold with its subjective component, the uncritical interpretation of the results can lead to erroneous conclusions. The visualization of speckles with antibodies against SC-35, a protein clearly present in speckles, provides a case in point. Using EFM, Fay et al. (1997) investigated the nuclear distribution of the signal generated by indirect immunofluorescent detection of an antibody against a phosphoepitope of the related splicing factors, SC-35 and SF2/ASF (Fay et al., 1997). As expected they observed a speckled pattern, yet also detected that 70–80% of their signal was found as a diffuse nuclear signal. Most of the latter, however, was not just background but apparently represented the SC-35 location in PFs in the PR (Spector et al., 1991), where cotranscriptional splicing occurs. Simple removal of this ‘‘background’’ would have supported the erroneous conclusion that SC-35 is a factor specifically contained in speckles but not elsewhere. Still, one may wish to count the number of speckles which remain as distinct clusters after eliminating ‘‘background’’ signal. If one does so, one should keep in mind that a fraction of the smaller speckles may not correspond to the interchromatin granule clusters defined by EM studies but rather represents aggregates of closely adjacent PFs. This example should just set a warning light that the choice of a threshold, which is not based on objective observer-independent criteria but rather on the experience, not to say the prejudice, of an investigator can lead to wrong interpretations. As a primer for beginners: the conclusion that colocalization

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of two proteins is proven when indirect fluorescence antibody staining of the two proteins in green and red color results in an image with yellow overlap is not considered state-of-the-art, although such evidence is widely published. The choice of a threshold procedure applied for removal of background must be carefully justified and documented. A threshold may be set for an entire observation field or various thresholds may be used for different parts of the field. The latter approach needs a clear justification. This, for example, is the case when background fluorescence is very inhomogenously distributed and much higher in some part of the observation field than in another. For a valid interpretation it is important to consider the choice of an appropriate threshold and other parameters of the imaging procedures in the light of as much information as possible about the biochemical nature of visualized nuclear domains, especially when they are considered as novel. The validity of the interpretation stands or fails with the specificity of the employed antibodies used and may be complicated by the dynamic participation of a given target proteins in different structures. Beyond the microscopic information per se, all this knowledge is an indispensable part of the interpretation of imaging data. Needless to say that background is also an important problem in EM studies. When performing a quantitative EM analysis, for example, the quantitative evaluation of immunogold labeling on ultrathin sections, one has to analyze the labeling density on resin outside the cells or tissue material as a reference for background signal.

5.3. Far-field fluorescence microscopy with resolution beyond the Abbe limit The optical resolution of a conventional far-field light microscope is given by the ‘‘Abbe/Rayleigh limit’’ (Abbe, 1873; Rayleigh, 1903), corresponding to roughly half the wavelength in the lateral direction and one wavelength in the axial direction. Recently, new laseroptical superresolution instrumentation has made it possible to extend the spatial analysis of cells far beyond the resolution limit of conventional FM (Table 1.1). This breakthrough does not invalidate the Abbe/Rayleigh theory of light microscopic resolution, which still describes an unsurpassable limit for conventional light microscopes. It shows, however, the limitation of this theory, which does not hold for certain laseroptical approaches which make use of possibilities beyond the technical limitations and imagination of Abbe’s time. This breakthrough has opened unprecedented opportunities for cell imaging (Cremer et al., 2006, 2010; Hell, 2009). The limit of light optical resolution is no longer given by the wavelength of the light used to generate an image, but by photon statistics: in order to resolve a structure with dimensions much below the wavelength of the fluorescent light emitted by a given structure with nanoscale dimensions (microscopy), it is necessary to record a sufficient number of photons from the object. The precision with which the position and hence

Table 1.1 Current methods of light optical nanoscopy Light optical nanoscopy

SALM PALM FPALM PALMIRA RESOLFT SPDM SPDMphymod ¼ RPM

Spectrally assigned localization microscopy Photoactivated localization microscopy Fluorescence photoactivation localization microscopy PALM with independently running acquisition Reversible saturable optical fluorescence transitions (ground state depletion microscopy) Spectral precision distance (or position determination) microscopy

SMIM

SPDM with physically modified fluorophores ¼ reversible photobleaching microscopy Dual color localization microscopy Structured illumination microscopy/patterned excitation microscopy Spatially modulated illumination microscopy

STED

Stimulated emission depletion microscopy

STORM, direct STORM

Stochastik optical reconstruction microscopy (subdiffraction resolution fluorescence microscopy) 4Pi (4p)-microscopy

2CLM SIM/PEM

4Pi

References

Betzig et al. (2006) Hess et al. (2006) Andresen et al. (2008), Geisler et al. (2007) Hell (2007, 2009) Bornfleth et al. (1998), Cremer et al. (1996, 1999), Edelmann et al. (1999), Esa et al. (2000, 2001), Heilemann et al. (2002) Baddeley et al. (2009b,c), Reymann et al. (2008), Lemmer et al. (2009) Gunkel et al. (2009) Frohn et al. (2000), Gustafsson et al. (1999, 2005, 2008), Heintzmann and Cremer (1999) Baddeley et al. (2007, 2009a), Failla et al. (2002a,b, 2003), Spoeri et al. (2004) Hell (2003, 2009), Dyba and Hell (2002), Hell and Wichmann (1994), Klar et al. (2000), Schmidt et al. (2008), Schrader et al. (1995) Heilemann et al. (2008), Rust et al. (2008), Steinhauer et al. (2008) Cremer and Cremer (1978), Hell et al. (1994), Hanninen et al. (1995)

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the distance between individually resolved neighboring objects can be measured is thought to be a better criterion to express optical resolution (Albrecht et al., 2001; Van Aert et al., 2006). It is clear that this problem is particularly severe in the case of live-cell imaging. Depending on the dynamics of a recorded structure with respect to changes in shape and position, it may become impossible to record sufficient photons at individual time points. Understandably, the new approaches with Abbe limit breaking resolution described below, typically require fixed cells, although it has become possible to develop approaches which overcome the Abbe/Rayleigh resolution limit for live-cell imaging as well. 5.3.1. 4Pi confocal laser scanning microscopy Using two opposing high numerical aperture (NA) lenses to concentrate two opposing laser beams constructively in a joint focus, confocal laser scanning 4Pi-microscopy (Baddeley et al., 2006; Egner et al., 2002; Hanninen et al., 1995; Hell et al., 1994) has become an established ‘‘optical nanoscopy’’ method, allowing an axial optical resolution down to the 100 nm regime, that is, about 6–7 times better than in conventional CLSM. The principal idea behind 4Pi-microscopy is to narrow the diameter of the focused laser beam at least in one dimension below the values possible if the beam is focused from one side only (as it is the case in a conventional confocal microscope). Such a procedure might be called ‘‘Point Spread Function Engineering.’’ Conventional lenses can possibly be replaced in the future by other means to focus light to a very small spot size. In the 1970s, C. Cremer and T. Cremer proposed a laser scanning microscope with improved optical resolution based on the use of a ‘‘4p point hologram’’ as focusing element. It was assumed that such a hologram might be generated using a point-like source with a diameter much below the wavelength of the light emitted from such a source or produced according to numerical calculations (Cremer and Cremer, 1972, 1978). This hologram with its capability to focus laser light from all sides should become the decisive optical element of a laser scanning microscope. A structure with dimensions above the point-like illumination spot could then be analyzed by ‘‘point-by-point’’ imaging of the fluorescence excited in this structure by the illumination spot. The possible optical resolution of this approach would depend on the smallest focal dimensions, which can be realized by the 4Pi point hologram. An ideal 4Pi point hologram might be approximated by a number of plane point holograms. Even in case that the spherical angle realized would be still considerably smaller than 360 , this new type of a scanning microscope may generate a smaller focus in all three dimensions as compared to microscopes equipped with one or two opposite conventional objectives. Considering the theoretical limitations of holographic focusing, one has to take into account that in the immediate vicinity of a light source much smaller

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than the wavelength of the light radiated by this source, effects such as the polarization properties of the electromagnetic field start to play a dominant role. In the case that a hypothetical spherical wavefront of constant intensity is focused from all sides (‘‘4Pi geometry’’) in a medium of constant refraction index, theoretically a spot diameter of about 1/3 of the wavelength may be obtained (Hell, 2007). Hence, the optical 3D resolution would be improved by a similar factor. For an excitation wavelength of 488 nm and a refraction index of 1.4, a spot diameter of about 1/3 of the wavelength means a limiting isotropic spot size around 100 nm. In contrast to the observation volume achieved by a conventional CLSM (200  200  600 nm3), this would mean a 24 times smaller observation volume (100  100  100 nm3) and a correspondingly improved 3D resolution. A pertinent example of the use of 4Pi microscopy was reported by Bewersdorf et al. (2006). These authors studied the topography of DSB repair and noted that H2AX exists in distinct chromatin structures. Focusing by a spherical wavefront of constant intensity and linear polarization is just one of the many possible illumination conditions. Arguably, this estimate may not provide the theoretical limit of holographic focusing. For example, using a radially polarized laser beam, the experimentally observed spot size for an NA ¼ 0.9 was observed to be about 35% below the theoretical limit for linearly polarized light; using specific photosensitive layers, even smaller spot sizes can be reached (Dorn et al., 2003). Similar effects should be obtained if the objective lens used in these experiments for focusing would be replaced by an appropriate plane point hologram. Instead of a single point hologram, in the 4Pi case 3D assemblies of plane holograms may be considered, where each single hologram is designed to contribute to the formation of a focus much below the dimensions, which can be achieved with conventional optics. Pulsed laser light with different wavelengths, including novel possibilities of attosecond laser physics (Silberberg, 2001) might be used to generate the appropriate spatial, temporal, and phase distribution of the illuminating field necessary to generate the desired focal point or pattern. Holograms might be designed, for example, to replace conventional optics with long working distances in laser scanning microscopes. The distance of the focus generated by a given hologram arrangement can be much larger than possible with any conventional system of lenses without compromising the desired characteristics of the focus. This advantage makes it possible to develop assemblies of holograms as new focusing devices from all directions of the space with geometries and hence spatial light distributions which are impossible to realize with conventional high NA objective lenses. The present state of 4Pi-microscopy provides a point in case. While it is possible to align two high NA objective lenses opposite to each other in order to generate the interference pattern necessary to obtain an increased optical resolution, the geometry of these objectives precludes the simultaneous use of additional high-aperture objectives at many different angles.

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Such a limit does—at least in principle—not exist for a 4Pi scanning microscope based on an assembly of holograms. Considering the possibilities and technical limitations to develop 4Pi holographic focusing devices instead of conventional optics, we feel presently not able to come to a safe conclusion regarding the technical limitations of ‘‘4Pi-focusing’’ with the purpose of generating a scanning-based fluorescent image with improved optical resolution. In principle, the smaller the exciting spot diameter, the better the effective optical resolution. We believe that the possibilities of 4Pi holographic focusing devices have not been fully explored and deserve further theoretical and experimental studies. Below, we consider stimulated emission depletion (STED) microscopy as a most elegant and successful approach to experimentally achieve a focal region with dimensions much below the Abbe limit. It might become possible to establish 3D STED microscopes equipped with holograms instead of conventional optics. 5.3.2. Stimulated emission depletion microscopy STED microscopy was first conceived and realized in the 1990s (Hell and Wichmann, 1994; Schrader et al., 1995). The basic idea of STED microscopy is to reduce the size of the region of a biological object, such as a cell, in which fluorescence is excited by a very short excitation pulse. This goal is achieved by a ‘‘depletion’’ pulse (‘‘STED pulse’’), which follows immediately after the excitation pulse and acts in the vicinity of the center of the fluorescent region. This is done in such a way that the STED pulse forms a ring around the center of the fluorescent region. As a consequence, fluorescence is only detected from a much smaller region. Due to the scanning mechanism, the position of this smaller fluorescent region can be identified with an accuracy down to the 1 nm range. Accordingly, the recorded fluorescence signal can be assigned to this smaller region further improving the optical resolution. STED microscopy was the first successful implementation of more general concepts of focused beam superresolution approaches like RESOLFT (REversible Saturable OpticaL Fluorescence Transitions) or ‘‘ground state depletion microscopy’’ and has already presently found numerous applications in high resolution cell biology (Donnert et al., 2006; Hell, 2007). In principle, there is no limitation with respect to the lateral resolution, which can be obtained by STED microscopy except for the requirement that an amount of photons must be recorded from any given illumination point sufficient to generate an image. Using a combination with 4Pi-microscopy, STED has recently been realized also in three dimensions with a 3D resolution in the lower nanometer range (Schmidt et al., 2008). 5.3.3. Structured illumination microscopy (SIM) Improvements of light-optical resolution beyond the Abbe/Rayleigh limit achieved in the case of 4Pi confocal microscopy and STED microscopy became possible by the application of ‘‘just physics.’’ This means that in principle a better resolved image is obtained without a need for further

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complex data processing. Other modes of circumventing the Abbe/Raleigh limit have been implemented by a combination of advanced optical and computational methods. One of these additional possibilities is to illuminate the object with an appropriate pattern of light; moving either the object or the pattern, at each relative position an image of the object is taken by a highly sensitive CCD camera. Using complex but well-established algorithms in the Fourier space, from such images it becomes possible to reconstruct an image of the object at enhanced effective optical resolution (Frohn et al., 2000, 2001; Gustafsson, 2005; Gustafsson et al., 1995, 1996; Heintzmann and Cremer, 1999; Schermelleh et al., 2008). Recently, even a 3D optical resolution enhancement and its application to the analysis of structures at the nuclear envelope has become possible (Fig. 1.9). 5.3.4. Spatially modulated illumination microscopy (SMIM) SMIM (Baddeley et al., 2007; Failla et al., 2002a,b, 2003; Spoeri et al., 2004) is another possibility to use structured illumination to improve spatial analysis. It is based on the creation of a standing wave field of laser light (Bailey et al., 1993). This can be realized in various ways, for example, by focusing coherent light into the back focal planes of two opposing objective lenses of high NA. The fluorescence-labeled object is placed between the two lenses and moved axially in small steps (e.g., 20 or 40 nm) through the standing wave field. At each step fluorescence is registered by a highly sensitive CCD camera (Fig. 1.10). This procedure allowed to measure the diameter of individual fluorescent objects down with sizes of about 1/15 of the exciting wavelength, that is, down to a few tens of nanometers for visible light, and to determine axial distances between ‘‘point-like’’ fluorescent objects at the lower nanometer scale with a precision in the 1 nm range (Albrecht et al., 2002). Several biophysical application examples indicated the usefulness of SMI-‘‘nanoscopy’’ for the study of the size of individual chromatin regions (Hildenbrand et al., 2005; Mathee et al., 2006; Reymann et al., 2008) and of TFs (Martin et al., 2004). In the latter case, the SMI results obtained were comparable with EM images obtained from the same type of specimens, revealing mean size differences in the 30 nm range. In other SMI-nanoscopy experiments, high throughput precision size measurements of replication foci were performed (Baddeley et al., 2009a). 5.3.5. Spectral precision distance/position determination microscopy (SPDM) SPDM and related techniques (Bornfleth et al., 1998; Cremer et al., 1996, 1999, 2002; Heilemann et al., 2002, 2004; Lacoste et al., 2000; Schmid et al., 2000; van Oijen et al., 1999) are far-field FM approaches based on labeling of neighboring ‘‘point-like’’ objects with different spectral signatures, spectrally selective registration, and high precision position monitoring, that is, a method of ‘‘spectrally assigned localization microscopy’’ (SALM). Combined with careful calibration of optical aberrations, this

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5 Phases for each z-section

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Figure 1.9 Structured illumination microscopy (SIM) in three dimensions. Top: Scheme of 3D-SIM: by using a diffraction grating, an illuminating sine wave pattern is produced in the object space. At each axial (z) position, five different phases of the sine wave pattern are recorded. Three image stacks are registered with the diffraction grating sequentially recorded into three positions 60 apart. This allows to calculate a 3D-SIM image with enhanced details. Bottom: Comparison of a deconvolved light optical nuclear midsection obtained with a conventional laser confocal scanning microscope (left) and a light optical midsection obtained with 3D-SIM (right). Simultaneous imaging of DNA, nuclear lamina, and nuclear pore complex (NPC) epitopes was performed in C2C12 cells following labeling with antibodies against lamin B (green) and NPC epitopes (red). DNA (blue) was counterstained with DAPI. Enlargements of the periphery of the whole 3D SIM nuclear image suggest channels starting at nuclear pores and permeating through the lamina and between chromatin clusters (cf. Fig. 1.4). These details are not recognizable in the confocal midsection. From Schermelleh et al. (2008), reprinted with permission from AAAS.

allows the measurement of positions and mutual distances between ‘‘pointlike’’ fluorescent objects in a range far below the ‘‘Abbe limit’’ of distance resolution for objects of the same spectral signature ( 200 nm). ‘‘Proof-of-principle’’ examples for the application of SPDM in nuclear genome structure research have been the analysis of the BCR-ABL region correlated with chronic myeloid leukemia (Esa et al., 2000); of conformational differences in the 3D-nanostructure of the immunoglobulin heavychain locus, a hotspot of chromosomal translocations in B lymphocytes (Esa et al., 2001); of the nanostructure of imprinted gene domains in human

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Figure 1.10 Spatially modulated illumination microscopy. Top: Scheme of SMI (spatially modulated illumination) microscopy. A laser beam is focused from two sides into the back focal planes of two opposing high NA objective lenses, producing between them a standing wave field. The object is moved along the optical axis within this field in precise steps, for example, 20 or 40 nm. At each step, the fluorescence image excited by the laser beam is registered by a highly sensitive CCD camera. Bottom: The size (extension along the optical axis) of RNA Pol II sites in the nucleus of HeLa cells was measured by SMI microscopy, and by electron microscopy (EM) using cryosections of ca. 140 nm thickness. One hundred and forty-eight sites were measured by SMI and 105 by EM. The abscissa gives the measured SMI/EM sizes in nanometers. The average size determined by SMI was 74 nm, and the average size by EM was 45 nm. Reproduced from Martin et al. (2004), with permission from American Society for Cell Biology.

interphase nuclei (Rauch et al., 2000, 2008); or of the distribution of genes in the active and inactive X-CT (Dietzel et al., 1999). In these early SPDM applications, differences in the fluorescence emission spectra were used as spectral signatures. Generally, SPDM (SALM) requires that in a given observation volume (defined, e.g., by the full-width-at-halfmaxima (FWHM) of the Point Spread Function of the microscope system used), there is just one object with a given spectral signature to be measured at a given time, a condition referred to as ‘‘optical isolation.’’ Since the reasonable

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number of useful different emission spectrum-based signatures is still limited (presently 7), this means that clusters of numerous fluorescent molecules of the same type (e.g., 400 molecules in an observation volume of 0.2  0.2  0.6 mm3) cannot be resolved in this way. The use of differences in the emission spectra, however, is only one of the many ways to realize the ‘‘photon sorting’’ required for the localization of just one molecule in the observation volume at a given time: already in the original SPDM concept, spectral signatures were conceived to include also other ‘‘photon sorting’’ modes like fluorescence lifetimes, photoluminescence, and stochastic-labeling schemes to allow photophysical discrimination (Cremer et al., 1996, 2002). To realize such ‘‘monocolor’’ spectral signatures, ‘‘proof-of-principle’’ experiments have been performed on the basis of fluorescence lifetimes to measure localizations of single molecules and the distance between them in a range down to few tens of nm (Heilemann et al., 2002, 2004). In this way, SPDM measurements allowed distance determinations too large for FRET techniques but considerably below the optical resolution of conventional, confocal, or 4Pi-microscopy. Since then, a number of conceptually related SALM methods have been described, such as BLINKING, FPALM, PALM, PALMIRA, SMACM, STORM, dSTORM etc. (Betzig et al., 2006; Biteen et al., 2008; Geisler et al., 2007; Heilemann et al., 2008; Hess et al., 2006, 2007; Huang et al., 2008; Juette et al., 2008; Lidke et al., 2005; Rust et al., 2006). Single molecule microscopy has become possible by CCD cameras with sensitivity sufficient for the routine imaging of single fluorochromes. This method has already been applied to trace structures labeled with a single fluorochrome in living cells (Grunwald et al., 2008; Lange et al., 2008; Seisenberger et al., 2001). Although in various SALM methods, the lateral (x,y) localization of single molecules (object plane perpendicular to the optical axis) has been firmly established, the problem of localization along the optical axis (z) has proven to be challenging. To obtain 3D reconstructions of the labeled objects (i.e., the x,y,z coordinates of the fluorescently labeled molecules), various approaches have been applied. One solution is to use confocal laser scanning or confocal laser scanning 4Pi-microscopy (Esa et al., 2000; Rauch et al., 2000, 2008; Schmid et al., 2000; Hueve et al. 2008) to obtain the 3D positions of the objects. Another possibility is based on the 3D information within the laterally acquired signal. Since all light-emitting molecules are ‘‘point-like’’ compared with the wavelength used, one can assume that they all are imaged in the same way. The fact that out-of-focus objects appear more blurred and using a PSF which is not symmetric along the optical axis can be used to localize photon-emitting sources in all spatial dimensions. If the propagation path of the electromagnetic waves is well known, under ideal registration conditions the accuracy of the axial SMI localization (z) is restricted only by the number of photons detected (Albrecht et al., 2001), analogous to other microscopy types of localization (Cremer et al., 1999). Using common photoactivatable or photoswitchable fluorophores in

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combination with biplane detection ( Juette et al., 2008) or a systematically modified detection PSF (Huang et al., 2008), a 3D single molecule localization accuracy of about 60–80 nm FWHM was recently achieved. Recently, it was demonstrated that using SPDM with physically modifiable fluorochromes (SPDMphymod) conventional fluorochromes such as Alexa 488 or Alexa 568 and 647 (Baddeley et al., 2009a,b; Lemmer et al., 2009; Reymann et al., 2008), the green fluorescent protein variant YFP (Lemmer et al., 2008), or other standard variants (GFP, mRFP) can be used for nanoimaging of cellular nanostructures (Fig. 1.11) (Gunkel et al., 2009). Here, dual color localization microscopy (2CLM) is used under conditions, which result in ‘‘reversible photobleaching’’ of the respective fluorophores (Kaufmann et al., 2009; Lemmer et al., 2008; Reymann et al., 2008). In principle, this means switching of the molecules from a ‘‘bright’’ fluorescent state into a metastable ‘‘dark’’ state (Patterson and Lippincott-Schwarz, 2002; Sinnecker et al., 2005). From this state they return to the ‘‘bright’’ state by a stochastic process, which can be described by fluorescent lifetimes

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Figure 1.11 Comparison of conventional epifluorescence microscopy and nanoimaging of mRFP-tagged histones H2A (red) and GFP-tagged Snf2H transcription factors (green) within a human U2OS nucleus (Gunkel et al., 2009). (A) Conventional epifluorescence image. (B) 2CLM image, here the position of individual mRFP-tagged H2A histones and GFP-tagged Snf2H transcription factors is visualized as a single dot with a size representing the individual localization accuracy. (C)–(E) display Enlargements from the boxed regions in (A) and (B), respectively. Note that the magnification of the conventional image is empty, that is, it does not provide better resolved structural details in contrast to (D) and (E). Scale bars are 2 mm in (A) and (B), 500 nm in (C) and (D), and 100 nm in (E).

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of the ensemble in the order of several seconds. Simple considerations in analogy to radioactive decay show that such large lifetimes allow highly resolved localization microscopy imaging (Cremer et al., 2010). In contrast to SALM methods such as FPALM, PALM, or STORM based on two frequency photoswitching (Betzig et al., 2006; Hess et al., 2006; Huang et al., 2008), only one laser line per fluorochrome type is required to induce fluorescence/luminescence lifetimes in a given fluorochrome on the second timescale. The burst (or ‘‘flash’’) of photons, which is emitted from a single fluorophore, allows to determine its position with nanometer accuracy under the condition of optical isolation, that is, the conditions must be such that all other similar fluorophores, which are located closer to the emitting fluorophore than the Abbe limit, should not emit photons of the same spectrum at the same time. We recently showed that reversible photobleaching can be achieved by using an excitation intensity in the 10 kW/cm2 to 1 MW/cm2 range (Baddeley et al., 2009). For example, Fig. 1.11 shows chromatin visualized by mRFP-tagged H2A together with the location of single GFP-tagged ATPase subunit Snf2H molecules (Gunkel et al., 2009), which define a certain class of chromatin remodeling complexes (Becker and Horz, 2002; Cairns, 2007; Rippe et al., 2007). The results indicate the feasibility to reveal details on the interaction of remodeling complexes with chromatin at unprecedented effective resolution. SPDM in combination with SMI along the optical axis is a further method to achieve 3D localizations of individual molecules and a corresponding 3D effective optical resolution. Using appropriately labeled cellular structures, a lateral effective optical resolution of 10–20 nm was realized together with an axial effective optical resolution around 30–50 nm. Thus, an overall 3D effective optical resolution around 40–50 nm was achieved, corresponding to about 1/10 of the wavelength used (Lemmer et al., 2008). In summary, novel developments in laseroptical nanoscopy will reduce the gap in resolution between ultrastructural methods (Angstroms to a few nanometer resolution) and visible light far-field microscopy (conventionally hundreds of nanometer resolution). 5.3.6. Perspectives for in vivo imaging at the nanometer scale Laser optical nanoscopy has typically been performed on fixed cells in order to demonstrate the achievable optical resolution. Although the application of these techniques to live-cells imaging is still in its beginning, some preliminary results indicate its feasibility. For example, Westphal et al. (2008) analyzed the movement of fluorescently labeled synaptic vesicles in living cells recorded with STED microscopy at a rate of 28 frames/s and an optical resolution around 60 nm laterally. In this study the cross-section area of the focal STED spot was reduced about 18-fold below the conventional diffraction limit of about 260 nm. In another application (Nagerl et al.,

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2008), STED microscopy was used for live-cell imaging of dendritic spines to dissect synaptic vesicle movement at video-rates. Due to the scanning mechanism, high-speed nanoimaging in STED requires small regions of interest (in the few micrometer range). In contrast, the various modifications of SALM have the potential for in vivo nanoimaging of large cellular areas (up to 100  100 m2; in combination with new optical techniques, even larger ones). For example, Hess et al. (2007) used FPALM to study the dynamics of hemagglutinin cluster distribution in membranes of living cells at 40 nm effective optical resolution to discriminate between raft theories. Preliminary experiments have demonstrated that live-cell imaging is also possible using SPDM with a physically modified fluorophores (SPDMphymod) approaches: In live human cells, the distribution of fluorescent-labeled membrane proteins was registered at an effective optical resolution of few tens of nanometers (Y. Weiland, P. Lemmer, C. Cremer, unpublished observations). In the SPDMphymod experiments, a CCD camera was used allowing to register 15–20 frames/s. The use of high-speed, back-illuminated CCD cameras allowing to register up to 1000 frames/s will highly extend the possibilities of this approach in live-cell imaging applications.

5.4. Importance of correlation microscopy for new ways to realize an old concept As described above, microscopic analyses of living or fixed cells have their own advantages and limitations, including the possibility of artifacts, which may result from necessary pretreatments of cells prior to microscopy and from limitations of the chosen microscopic method itself. The important point is that available FM and EM approaches are complementary in different ways. Therefore, for a comprehensive, sequential investigation of the same cell, these techniques can and should be combined to add up the advantages and to compensate for the disadvantages of the exclusive use of each method. Correlative microscopy based on the possibility to identify the same structure and its position first with one type of microscopic approach and then with another has opened the way for a wide spectrum of novel applications (Albiez et al., 2006; Karreman et al., 2009; Sartori et al., 2007). Let us consider correlative microscopy to reveal the structure (or structures) of a nuclear compartment or domain. Ideally, such studies should start with a living cell with the intent to follow the fate of such a domain over a period of time. After the application of a fixation protocol suitable to preserve the in vivo 3D arrangement present immediately before fixation as best as possible, the same cells are reinvestigated sequentially by superresolution FM (with resolution beyond the Abbe limit) and finally by EM. The comparison of structural details seen in the living and the fixed state of the same cell allows an investigator to analyze the same nuclear domains of an individual cell. In addition, one can appreciate structural changes that may occur during the

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application of preembedding strategies to visualize distinct nuclear constituents. Unfortunately, preparative procedures for FM observations often alter the fine nuclear structure, thus precluding further EM analysis. Consequently, the combination of living cell FM and subsequent EM investigation presently remains the best approach (Albiez et al., 2006). The optimal use of the concept of correlative microscopy for studies of nuclear architecture will in many cases require the generation of both FM and EM 3D images. While 3D reconstructions based on stacks of ultrathin sections are possible, one must keep in mind that it is quite demanding to make tens or even hundreds of consecutive sections without losing sections or section order. If one wishes to make serial sections from biological specimens much thicker than a single cell, for example, from entire mammalian preimplantation embryos, the number of consecutive sections required to obtain a full 3D reconstruction can quickly go up to thousands, while 3D reconstructions based on hundreds of consecutive light optical sections can routinely be obtained at the FM level. For a solution, one can combine ultramicrotomy with scanning EM (SEM) (Denk and Horstmann, 2004). Here, the surface of the specimen is imaged, an ultrathin section is removed by the ultramicrotome, the remaining surface is scanned again, and so forth (e.g., see Fig. 1.3). Whereas consecutive sections are the essential material for TEM, they play no role for imaging and are thrown away. This approach was applied for the first time, in combination with a selective preembedding staining of DNA, to reconstruct in 3D intranuclear distribution of chromatin and to quantitatively evaluate the part of the nuclear volume occupied by chromatin and by the chromatin-poor/-free interchromatin space (Rouquette et al., 2009). Alternatively, SEM can be combined with focused ion beam (FIB) milling (Knott et al., 2008) of the specimen. Here, an ion beam removes very thin layers of material (down to 10 nm), thus improving the z resolution. Schroeder-Reiter et al. (2009) used this approach for the first time to study the 3D ultrastructure of chromosomes. Compared with TEM, the resolution limit of SEM is lower but still considerably better than the resolution, which can presently be obtained with the most advanced superresolution FM microscopes overcoming the Abbe limit. The largest possible observation surface is much smaller with the FIB/SEM method (areas of about 20  20 mm2) compared to the ultramicrotome/SEM method (up to several hundred micrometers). While both methods hold the promise to carry out 3D EM reconstructions from very large numbers of consecutive SEM images in a routinely applicable way, one should keep in mind that these two methods strictly require preembedding approaches with all the qualms concerning the typically less optimal structural preservation compared with postembedding approaches. Despite this limitation we expect that 3D EM reconstructions based on large series of consecutive SEM images can make important contributions to a future correlative microscopy, where single cells are followed from the living to the fixed state combining a wide range of FM and EM methods. In addition to SEM the use of TEM in cryoelectron microscopy can be considered for 3D studies using tomographical methods (Medalia et al., 2002), but

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the application seems for the moment limited to whole nuclei in the studies of NPCs (Beck et al., 2004). Protocols proposing in vivo introduction of probes into the cells or directly into nuclei, suitable for both FM and EM analysis, may be another useful tool for future investigations enabling both FM and EM observations in successive steps on the same sample (Kireev et al., 2008).

6. Concluding Remarks We present a brief historical account of light and electron microscopic studies on nuclear structure followed by an overview of current models of the functional nuclear architecture and of experimental support obtained with microscopic methods. We review present evidence for the intranuclear location of sites, where major nuclear functions such as DNA replication and repair, transcription, and RNA transport take place, focusing in particular on the topographical and structural implications of gene expression. In close relation to this point, we tried to evaluate the strength and the degree of reliability and reproducibility of different microscopic approaches that gave rise to such hypotheses and models. Due to its superior resolution EM provided first evidence for a complex chromatin organization and—in combination with DNA-specific staining procedures—for a largely DNA-free interchromatin space carrying interchromatin granules and various nuclear bodies. In particular, EM was instrumental for revealing the PR as the major subcompartment for DNA replication, hnRNA synthesis, cotranscriptional splicing, and nucleotide excision DNA repair. FM combined with in situ hybridization (FISH) and immunocytochemistry provided direct evidence for CTs and their substructure, including chromatin domains with various patterns of histone modifications, as well as chromatin arrangements down to the positioning of individual genes. We then point out problems and open questions in the field of functional nuclear architecture and discuss possibilities to answer them with emphasis on current limitations and new options of quantitative microscopic analyses. A great effort is being made in developing or perfecting light and electron microscopic techniques with the goal to follow cellular processes under the best experimental and methodological conditions. Compared with FM, EM has the decisive advantage of its highly superior resolution. The possibility to discriminate specific nuclear structures by multicolor approaches and the easiness to obtain light optical serial sections for 3D image reconstructions provide major advantages of CLSM. Spinning disk confocal microscopy is presently the most promising approach for 4D (space and time) studies of the dynamic nuclear organization in living cells. The introduction of in vivo markers for vital fluorescence microscopic analyses constitutes a major contribution and is closely related to advancements of molecular biological studies.

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New types of FM have become available for cell imaging with a resolving power far beyond the Abbe limit of conventional light microscopy. This breakthrough has been accentuated by the new terms, light optical nanoscopy or superresolution microscopy, notwithstanding the fact of the still far superior resolution limit of EM. Staining of nuclear structures in fixed cells sometimes includes harsh preparative conditions, such as a DNA denaturing step prior to in situ hybridization. Structural damage caused by such procedures compromises the advantage of improved resolution. The applicability of light optical nanoscopy for live-cell studies is an urgently awaited further breakthrough, because such an approach, besides its impact for high resolution studies of nuclear dynamics, would allow rigorous tests how certain fixation and postfixation procedures alter fine structural features present in the living cell. New 3D EM approaches combining SEM either with ultramicrotomy or with FIB milling also show great promise for novel achievements. Correlative microscopy consisting in successive observations by light microscopy in vivo followed by studies of the same cell after fixation with advanced light and electron microscopy is obviously the best approach to reveal the complex topography of major nuclear structures in 3D and 4D. It will help to capitalize on the particular strengths of each microscopic method and to avoid specific disadvantages. Major progress of our insight into the complex relationships between nuclear structure and function from dynamic higher order chromatin arrangements to the topography of protein machineries involved in transcription, splicing, DNA replication and repair, to topographical aspects of the nuclear import and export of macromolecules requires a system’s biology approach. For this purpose, methods of light and electron microscopy need to be combined with high-throughput methods for the mapping of DNA–DNA interactions in cis and trans, as well as DNA–protein and DNA–RNA interactions. Microscopic methods are a powerful tool in the investigation of structure–function relationships in the cell nucleus. We hope that this review will provide a good overview of different approaches and will constitute a sort of guide to scientists who look for the right microscopic methods to be applied to their research problem.

ACKNOWLEDGMENTS We are indebted to Ron Hancock (Laval University, Quebec, Canada) and Denise Sheer (University of London, Great Britain) for critical reading of early versions of the manuscript and helpful suggestions. The work of the authors’ laboratories has been supported by the Deutsche Forschungsgemeinschaft, the European Union, the Munich Center of Integrated Protein Science (CIPSM), the LMU Bioimaging Network (BIN), and the Swiss National Science Foundation.

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Meiotic silencing in Caenorhabditis elegans Eleanor M. Maine Contents 1. Introduction 2. Chromatin Regulation in the Germ Line 3. Repressive Mechanisms in C. elegans Meiotic Germ Line 3.1. Extrachromosomal transgenic arrays 3.2. X Chromosome silencing 3.3. Genome-wide analysis of germ line gene expression 3.4. Enrichment of H3K9me2 on male X and other unpaired chromosomes 3.5. Targeting mechanisms 3.6. Enrichment of H3K27me3 on X chromosomes 3.7. Regulation of germ line H3K27me3 by Polycomb Repressive Complex 2 (PRC2) system 3.8. Summary 4. Meiotic Silencing and Germ line Development in C. elegans 4.1. Repression of DNA insertions 4.2. Maintenance of genome integrity 4.3. Epigenetic control of embryogenesis 4.4. Transcriptional regulation of single X 4.5. Chromosome evolution 4.6. Summary 5. Mechanistic and Functional Comparison of Meiotic Silencing Phenomena in Different Species 5.1. Meiotic silencing in N. crassa 5.2. Meiotic silencing in insects 5.3. Meiotic silencing in mammals 5.4. Meiotic silencing in birds 5.5. Other meiotic transsensing phenomena in vertebrates 5.6. Summary 6. Noncoding RNA and Chromatin Structure

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7. Conclusions and Future Prospects 7.1. Mechanism 7.2. Function Acknowledgments References

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Abstract In many animals and some fungi, mechanisms have been described that target unpaired chromosomes and chromosomal regions for silencing during meiotic prophase. These phenomena, collectively called ‘‘meiotic silencing,’’ target sex chromosomes in the heterogametic sex, for example, the X chromosome in male nematodes and the XY-body in male mice, and also target any other chromosomes that fail to synapse due to mutation or chromosomal rearrangement. Meiotic silencing phenomena are hypothesized to maintain genome integrity and perhaps function in setting up epigenetic control of embryogenesis. This review focuses on meiotic silencing in the nematode, Caenorhabditis elegans, including its mechanism and function(s), and its relationship to other gene silencing processes in the germ line. One hallmark of meiotic silencing in C. elegans is that unpaired/unsynapsed chromosomes and chromosomal regions become enriched for a repressive histone modification, dimethylation of histone H3 on lysine 9 (H3K9me2). Accumulation and proper targeting of H3K9me2 rely on activity of an siRNA pathway, suggesting that histone methyltransferase activity may be targeted/regulated by a small RNA-based transcriptional silencing mechanism. Key Words: Meiotic silencing, Germ line, H3K9me2, Chromatin, RNA-directed RNA polymerase, Histone modification, X chromosome, RNAi. ß 2010 Elsevier Inc.

1. Introduction The term ‘‘meiotic silencing’’ refers to the silencing of unpaired/unsynapsed chromosomes and chromosomal regions during prophase of meiosis I. Meiotic silencing has been studied in many animal species (e.g., mammals, birds, nematodes, insects) as well as certain fungi (Baarends et al., 2005; Cabrero et al., 2007; Kelly et al., 2002; Mahadevaiah et al., 2009; Schoenmakers et al., 2009; Shiu et al., 2001; Turner et al., 2005). In animals, meiotic silencing is thought to include the silencing of sex chromosomes in the heterogametic sex, a process called meiotic sex chromosome inactivation (MSCI) (Handel, 2004). Subsequent to the discovery of MSCI, researchers used mutations and chromosomal rearrangements to examine the regulation of unpaired autosomes and found them to be regulated in a similar fashion. Hence, meiotic silencing appears to be a general process not unique to the heterogametic germ line or

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chromosomes. Meiotic silencing phenomena have different characteristics in different species, as their organism-specific names reflect. Collectively, these phenomena have been referred to as meiotic silencing (e.g., Kelly and Aramayo, 2007), although the functional relationships among them are not completely clear. Meiotic silencing in animals typically occurs at the chromatin level and involves accumulation of histone modifications that are thought to promote a closed chromatin configuration and transcriptional repression. In addition to transcriptional repression, these changes in chromatin structure may contribute to meiotic chromosomal events such as chromosome disjunction. In Caenorhabditis elegans, where failure of chromosomes to pair and synapse triggers accumulation of histone silencing modifications on the unpaired chromatin, the process is referred to as meiotic silencing of unpaired chromatin (MSUC; Maine et al., 2005). In mouse, the process is referred to as meiotic silencing of unsynapsed chromatin (MSUC; Schimenti, 2005) because the failure of chromosomes or chromosomal regions to synapse is the trigger for accumulation of histone silencing marks and transcriptional repression. A fundamentally different meiotic silencing mechanism is at work in the fungus, Neurospora crassa, where the presence of unpaired chromatin triggers silencing not only of that unpaired region but also of homologous paired DNA elsewhere in the genome. This phenomenon is termed meiotic silencing by unpaired DNA (MSUD; Shiu et al., 2001) and appears to occur strictly at a posttranscriptional level.

2. Chromatin Regulation in the Germ Line Meiotic silencing can be considered within the context of germ line development. Animal germ cells undergo specific differentiation programs to produce gametes that have the capacity, upon fusion, to give rise to a new individual. Hence, the chromatin in haploid gametes must have the flexibility to reorganize during early embryogenesis and support the development of diverse cell lineages. Germ line development requires mechanisms that allow the formation of gametes while also protecting germ cells from the expression of gene products that might decrease progeny viability. The misregulation of gene expression during germ line development can have disastrous consequences for fertility and for the health and development of offspring. One important mechanism of gene regulation in all tissues, including the germ line, is the modulation of chromatin structure to promote or repress transcription. Chromatin structure in the germ line must also accommodate special features of germ cell biology, including homolog pairing, synapsis, and recombination.

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The mechanisms of chromatin regulation have been discussed in several recent excellent reviews (e.g., Gelato and Fischle, 2008; Kouzarides, 2007; Rando and Chang, 2009; Wu et al., 2009) and will be discussed only briefly here. The basic unit of chromatin is the nucleosome, which includes DNA wrapped (twice) around an octamer of histone proteins. Chromatin is compacted to differing degrees, both locally and at the level of the entire chromosome. Chromosome condensation is critical for chromosome segregation during the mitotic and meiotic divisions. Local chromatin regulation modulates the ability of nonhistone proteins to contact the DNA and, thereby, regulate transcription. Intense effort has identified a large collection of conserved histone modifications that correlate with transcriptional states and are thought to alter chromatin structure, thereby promoting or preventing transcription (Kouzarides, 2007). Early studies identified certain modifications as associating with expressed or repressed chromatin based on indirect immunofluorescence labeling. For example, H3K9me3 was observed to be enriched in constitutive heterochromatin in mouse, Drosophila, and mealy bug cells, including at centromeres, some telomeres, the inactive X chromosome in female mouse cells, and the highly condensed X and Y chromosomes (together with associated factors referred to as the XY-body) in male meiotic germ cells (Cowell et al., 2002). More recently, genome-wide mapping studies have begun to provide detailed information about the fine-structure distribution of specific modifications (Barski et al., 2007; Kolasinska-Zwierz et al., 2009; Rando and Chang, 2009). In general, active genes tend to have a nucleosome-free region at the transcription start site. Some histone modifications (also called histone marks) such as trimethylation of histone H3 on lysine 4 (H3K4me3) are found on nucleosomes immediately flanking the transcription start site of active genes. Other marks such as H3K36me3 are found on nucleosomes within exons of active genes. In contrast, for silent genes, marks such as H3K27me3 are found on nucleosomes extending several kilobase pairs upand downstream of the transcription start site. Interestingly, although many modifications appear to be associated primarily with active or silent loci (e.g., H3K4me3 or H3K27me3, respectively), the correlations for other marks are not so clear, and many questions remain about how (or whether) specific histone modifications influence transcription. A situation directly relevant to the analysis of meiotic silencing is the fact that although the presence of certain histone marks correlates with increased or decreased transcription, it has been a challenge to show cause and effect in most cases. For example, it is not clear whether histone ‘‘activation’’ marks change chromatin structure in such a manner as to allow transcription or whether they arise as a consequence of transcription. The story is simpler at the level of phenotype in the sense that defects in the ability to make specific chromatin modifications in the germ line are known to reduce or

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eliminate fertility in a variety of species, as discussed below. Moreover, the inheritance of inappropriately modified chromatin can contribute to developmental defects and increased susceptibility to disease (Chong et al., 2007; Kimmins and Sassone-Corsi, 2005; Strome and Kelly, 2007; Turner, 2007; Zamudio et al., 2008). Unique aspects of chromatin biology in the germ line include not only meiotic silencing but also the chromatin reorganization that occurs at diagnostic times during development, imprinting of maternal or paternal alleles, and the extreme condensation of sperm chromatin via histone replacement by protamines (Allegrucci et al., 2005; Hajkova et al., 2008; Kimmins and Sassone-Corsi, 2005). This review focuses on the mechanism and function of meiotic silencing in C. elegans, and discusses this phenomenon within the larger context of meiotic silencing processes in general. Meiotic silencing is of special interest as a chromatin regulatory mechanism with functions in both the male and female germ lines in addition to its sexspecific roles in male germ line development.

3. Repressive Mechanisms in C. elegans Meiotic Germ Line C. elegans is a hermaphroditic species where the predominant sex, XX hermaphrodites, produce both sperm and oocytes. XX animals have a female soma and a germ line that is male (produces sperm) during larval development and becomes female (produces oocytes) at approximately the time of the molt preceding the adult stage. Males are XO and produce sperm; they typically result from fertilization of a nullo-X gamete (produced as a result of meiotic nondisjunction). This mode of sexual reproduction requires that XX and XO germ cells undergo spermatogenesis. The X chromosome receives different histone marks in XX versus XO germ lines, hence spermatogenesis can accommodate these different chromatin states. The organization of the mature gonad is shown in Fig. 2.1. The distal end of the gonad contains a population of proliferating germ cells that function like stem cells and are maintained in mitosis via signals from the somatic gonad. More proximally, the germ cells are arranged in sequential stages of meiotic prophase (leptotene–zygotene, pachytene, and diplotene), which can be distinguished based on nuclear morphology and analysis of stage-specific markers. Both males and hermaphrodites store mature, haploid sperm in the proximal gonad. Adult hermaphrodites contain growing in the proximal gonad. Oocytes have progressed to diakinesis stage by the time they are ovulated into the spermatheca and immediately fertilized by stored sperm.

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XX hermaphrodite

Proliferative region

Leptozygo

Pachytene Diplotene-diakinesis (growing oocytes)

Sperm

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Pharynx Proliferative germ cells

XO male Pachytene XX 1 sp

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Vulva

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Pharynx Sperm

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XO Proliferative region Sperm C

Proliferative Fan germ cells

Figure 2.1 Organization of the mature C. elegans germ line. Photomicrgraphs of dissected (A) hermaphrodite and (B) male adult gonads are shown. Tissue was fixed and stained with the DNA dye, DAPI. Proliferative germ cells are located at the distal end of each gonad arm. (A) Proliferating, leptotene–zygotene (lepto–zygo), pachytene, diplotene–diakinesis, and sperm nuclei are indicated. (B) Proliferating, leptotene– zygotene (lepto–zygo), pachytene, diplotene, primary spermatocyte (1
sp), and sperm nuclei are indicated. (C) Schematic representation of the hermaphrodite (XX) and male (XO) body.

Relatively few X-linked genes are expressed in the C. elegans germ line. In both male and hermaphrodite mitotic and meiotic germ cells, active RNA polymerase II (RNA pol II) is associated with autosomes and absent from the X chromosome, suggesting that few X-linked genes are expressed (Kelly et al., 2002). The presence of repressive marks on the X chromosome in both sexes correlates with gene expression profiling data indicating germ line expression of many autosomal genes but relatively little germ line expression of X-linked genes (Reinke et al., 2000, 2004). Ooi et al. (2006) obtained further evidence of X chromosome silencing in their analysis of histone H3 replacement during meiosis. Typically, the H3.1 isoform is replaced by H3.3 during transcription. H3.3 is not detected on the hermaphrodite X chromosomes until late pachytene stage and fails to appear on the male X at all (Ooi et al., 2006). The pattern of histone modifications in the germ line is consistent with all of the above observations (Kelly et al., 2002; Reuben and Lin, 2002). In both the XX and XO germ line, autosomes are relatively highly enriched for histone modifications associated with transcriptional activation, such as

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H3K4me2 (Kelly et al., 2002; Reuben and Lin, 2002). In contrast, germ line X chromosomes have relatively low levels of histone activation marks and are enriched for a mark that correlates strongly with transcriptional silencing, H3K27me3 (Bender et al., 2004). Superimposed on this regulation, during early pachytene stage the single male X becomes enriched for H3K9me2 (Kelly et al., 2002), a mark loosely correlated with silencing (e.g., see Barski et al., 2007; Rando and Chang, 2009). Transient H3K9me2 foci appear on (perhaps all) hermaphrodite chromosomes during a very narrow window in late meiotic prophase, but this process seems to be independent of the male X enrichment. To date, no other marks besides H3K9me2 and H3K27me3 have been reported to accumulate preferentially on the X chromosome in the C. elegans germ line although it is certainly possible that such marks exist. The general pattern of chromatin marks is diagrammed in Fig. 2.2 and summarized in Table 2.1. A detailed discussion of the observed chromatin modifications is presented below.

3.1. Extrachromosomal transgenic arrays The pattern of histone modifications in the C. elegans germ line was first investigated by researchers studying the phenomenon of transgene silencing (Kelly et al., 2002; Reuben and Lin, 2002). In C. elegans, transgenes are often produced via a method that results in production of highly repetitive, extrachromosomal arrays. DNA is injected into the germ line syncytium, where it does not readily integrate into an endogenous chromosome, but instead forms a long concatemer called an extrachromosomal transgene array (Stinchcomb et al., 1985). Extrachromosomal arrays are mitotically transmitted in a quasi-stable manner, perhaps due at least in part to the holocentric structure of C. elegans chromosomes. Genes present in highly repetitive arrays typically express in somatic tissues but often fail to express in germ cells (Kelly et al., 1997). Better germ line expression of transgenes is often observed when arrays are less repetitive, suggesting that the germ line silencing system is especially sensitive to repetitive sequences (Kelly et al., 1997). This silencing may be analogous to the silencing of centromere repeats and other repetitive DNA sequences that have been observed in other organisms. Using indirect immunofluorescence to detect specific histone modifications, Kelly et al. (2002) and Reuben and Lin (2002) demonstrated that germ line-silenced transgenic arrays lack H3K4me2 and are enriched for H3K9me2. Moreover, Kelly et al. surveyed several other histone modifications known to correlate with transcriptional activity, including H3K9/K14ac, H3S10phos, H4K8ac, and H4K16ac, and none was present on silent arrays. In contrast, histone activation marks were observed on a germ line-expressed transgenic array, consistent with the hypothesis that transgene expression depends on, or at least correlates with, chromatin state.

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Proliferating LeptoDiakinesis germ cells zygo Pachytene Diplotene (oocytes) Sperm

A XX

No MSUC H3K9me2 H3K27me3 MES silencing H3K4me2 X activation

X chromosome

H3K9me2 H3K27me3 H3K4me2

Autosomes Proliferating LeptoMeiosis germ cells zygo Pachytene Diplotene I and II

Sperm

XO H3K9me2 MSUC H3K27me3 MES silencing H3K4me2 No X activation

X chromosome

H3K9me2 H3K27me3 H3K4me2

Autosomes B

DNA

H3K4me2 Pachytene nuclei

DNA H3K9me2 Pachytene nuclei

Figure 2.2 The dynamic pattern of chromatin regulation in the germ line. (A) Schematic diagram of XX (upper) and XO (lower) germ cells as they enter and progress through prophase of meiosis I (see Fig. 2.1). Shaded bars indicate the relative level of three histone marks, H3K9me2, H3K27me3, and H3K4me2, on X chromosomes and autosomes. (B) Photomicrographs show H3K4me2 distribution in XX mid-pachytene nuclei and H3K9me2 distribution in XO mid-pachytene nuclei. In each image, DNA is labeled in red and the chromatin mark is labeled in green. Arrows indicate the X chromosomes. H3K4me2, a mark associated with actively expressed chromatin, is concentrated on the autosomes and not visible on the X chromosomes. Note that XO pachytene nuclei would have a similar H3K4me2 distribution at this time, which is prior to the late-pachytene X-linked gene activation observed in XX germ lines. H3K9me2, a mark associated with unpaired chromatin, is concentrated on the male X chromosome and barely detectable on the autosomes and hermaphrodite X chromosomes. H3K27me3, a mark associated with silent chromatin, is observed on X chromosomes and autosomes, but is particularly concentrated on Xs (adapted with permission from Strome and Kelly (2007). Copyright Cold Spring Harbor Laboratory Press).

This is supported by the observation that transgenes that are normally repressed in germ cells are activated in mutants with defective H3K27 methylation in the germ line (Holdeman et al., 1998; Kelly and Fire, 1998; Korf et al., 1998).

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Table 2.1 Histone modifications in the C. elegans germ line

Chromosome

Activation marks (mitosis; meiotic prophase)

Wild type Paired autosomes High Paired Xs Transient (hermaphrodite) Single X (male) Not detected Other unpaired chromatin High Unpaired autosomes and autosomal duplications Unpaired Xs Transient (hermaphrodite) Germ line-active High arrays Germ line-silenced Not detected arrays

H3K27me3 (mitosis; meiotic prophase)

H3K9me2 (meiotic prophase)

Low High

Low Low

Moderate

High

Low

High

High

High

NA

Low

NA

High

Indirect immunofluorescence was used to evaluate histone modifications. Relative intensity of labeling is indicated as high, moderate, low, or not detected. ‘‘Transient’’ indicates a brief period of labeling, as described in the text. NA, not assayed. Data are from Kelly et al. (2002), Reuben and Lin (2002), Fong et al. (2002), Bender et al. (2004), and Bean et al. (2004). Activation marks that were assayed include H3K4me2, H3K9/K14ac, H3S10phos, H4K8ac, and H4K16ac.

3.2. X Chromosome silencing The same indirect immunofluorescence approach revealed that the chromatin state of the X chromosome is different from that of autosomes throughout the germ line. Critical to these studies was the ability to distinguish the X chromosome from autosomes. Kelly et al. (2002) employed various methods to identify the X chromosome, including analysis of X chromosome::autosomal fusions, fluorescent in situ hybridization (FISH) with X-specific probes, and deconvolution microscopy. Reuben and Lin (2002) tentatively identified the X chromosome based on length, as it was known to be the shortest of the six C. elegans chromosomes. Both studies observed differential accumulation of histone activation marks within germ line nuclei. In mitotic and meiotic nuclei, a relatively high level of activation marks was consistently detected on autosomes. In contrast, activation marks were only obvious on paired (hermaphrodite) X chromosomes in late pachytene through diakinesis stages of meiotic prophase, and the single male X chromosome lacked activation marks altogether. Consistent with the pattern of histone activation marks, the activated form of

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RNA pol II was detected in association with autosomes but not with the X chromosome (Fong et al., 2002; Kelly et al., 2002). Analysis of histone modifications associated with transcriptional repression revealed a relatively high level of H3K9me2 marks on the male X, and a moderate enrichment for H3K27me3 on all (hermaphrodite and male) X chromosomes. In contrast, H3K27me2 does not appear preferentially associated with any particular chromosome (Bender et al., 2004; Kelly et al., 2002).

3.3. Genome-wide analysis of germ line gene expression X chromosome gene expression in the C. elegans germ line has also been explored using gene expression profiling and functional genomic analysis (Maeda et al., 2001; Piano et al., 2000, 2002; Reinke et al., 2000, 2004). Gene expression profiling has identified large sets of transcripts that are enriched in the germ line relative to the soma; these genes can be grouped into several categories based on expression pattern, including (i) germ lineintrinsic genes expressed in the XX and XO germ line, (ii) spermatogenesisspecific genes expressed in the XO and larval XX germ lines, and (iii) oogenesis-specific genes expressed in the female and adult hermaphrodite germ line (Reinke et al., 2000, 2004). These studies revealed that germ line-intrinsic and spermatogenesis-specific genes are underrepresented on the X chromosome relative to autosomes. In contrast, oogenesis-specific genes are not underrepresented on the X (Reinke et al., 2000, 2004), although essential ovary-expressed genes tend not to be X-linked (Maeda et al., 2001; Piano et al., 2000, 2002). A trend away from germ line-expressed genes on the X was borne out by subsequent genetic studies showing that, among sets of duplicated genes, those that are expressed in the germ line tend to be located on autosomes while those expressed in the soma may be located on the X chromosome (Maciejowski et al., 2005; Ohmachi et al., 2002). Taken together, these data are consistent with the pattern of H3K4me2 and other activation marks observed by Kelly et al. (2002) in the germ line: there is little X chromosome expression during mitosis, male meiosis, and spermatogenesis, but there is a burst of X-linked expression in oogenesis. In an initial attempt to determine the functional significance of the observed patterns of dynamic chromatin modifications, Kelly et al. (2002) compared the average transcript level for all genes versus oogenesisexpressed genes on each chromosome. They found that genes whose expression remains high during meiosis tend to be located on autosomes. In contrast, the average X chromosome transcript levels were two- to threefold lower than autosomal transcript levels in the germ line. In the soma, no significant difference in autosomal versus X chromosomal transcript level was observed. These data were consistent with X-linked transcription occurring in only a small subset of germ cells. Consistent with this hypothesis, when in situ hybridization analysis was used to visualize transcript

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distributions, X-linked transcripts were observed during the late-pachytene/ diplotene window (Kelly et al., 2002). For each gene examined, mRNA was first visible in late pachytene nuclei, consistent with the appearance of chromatin activation marks on the X chromosomes at that stage.

3.4. Enrichment of H3K9me2 on male X and other unpaired chromosomes Germ line H3K9me2 distribution is dynamic and chromosome specific (Kelly et al., 2002; Reuben and Lin, 2002). In XO nuclei, the single X chromosome does not have a pairing partner during meiosis. Goldstein (1982) had previously observed that the male X is highly condensed in pachytene nuclei and remains so through the rest of spermatogenesis. In the male, H3K9me2 marks are detected during pachytene–diplotene stages. The X chromosome contains a relatively high level of H3K9me2 marks while the autosomes contain a low (‘‘basal’’) level. In the XX hermaphrodite, H3K9me2 is not preferentially associated with the X chromosomes. Instead, a focus of H3K9me2 labeling is visible in early pachytene stage at the end of what may be an autosome (W. Kelly, personal communication). As nuclei progress to late pachytene, H3K9me2 foci are observed at many chromosomal sites (Kelly et al., 2002). These foci persist into diplotene stage before disappearing. Based on these data, H3K9me2 appears to have a major role in regulation of the single male X chromosome but a more limited role with respect to the pair of hermaphrodite X chromosomes. The relationship between these transient H3K9me2 foci and the H3K9me2 marks on the male X chromosome is unclear. Bean et al. (2004) later demonstrated that H3K9me2 can become enriched on autosomes and X chromosomes if they fail to pair in either sex. In their studies, Bean and colleagues took advantage of mutations that disrupt pairing and synapsis of homologous chromosomes. For example, they evaluated H3K9me2 labeling in him-8 mutants, where the hermaphrodite X chromosomes fail to pair and synapse. In him-8 hermaphrodites, H3K9me2 was enriched on both X chromosomes from early pachytene through diplotene stages, similar to what was observed for the male X. Hence, the disruption of pairing and/or synapsis appears to allow or trigger H3K9me2 accumulation. Bean et al. (2004) obtained consistent data when they evaluated the chromatin state of unattached (free) autosomal duplications, such as the unattached chromosome III duplications, sDp1, sDp2, and sDp3. In any given nucleus, a free duplication can remain unpaired or pair with an intact chromosome, forcing a portion of the intact homolog to be unpaired (see Fig. 2.3). Bean and colleagues detected one to two H3K9me2 foci in nuclei carrying a free duplication, and the timing of H3K9me2 accumulation was similar to that observed in him-8 mutants. These data are consistent with a mechanism that (i) senses the presence of unpaired/unsynapsed

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A Germline nucleus carrying sDp3

B Homologs fully synapsed

sDp3 sDp3 IIIP IIIM

IIIM

IIIP

Single H3K9me2 focus corresponds to sDp3 C sDp3 fully synapsed sDp3 IIIM

D sDp3 partially synapsed sDp3

IIIP IIIP IIIM

Single H3K9me2 focus corresponds to an intact homolog

Two H3K9me2 foci correspond to portions of sDp3 and a homolog

Figure 2.3 Inferred pairing and synapsis in nuclei carrying a chromosomal duplication. (A) Maternal (M) and paternal (P) copies of chromosome III are indicated, as well as the free (unattached) duplication, sDp3. sDp3 corresponds to approximately the left half of chromosome III. (B–D) Meiotic nuclei. Heavy line indicates H3K9me2 enrichment. (B) Maternal and paternal homologs are fully paired and synapsed. sDp3 is unpaired and unsynapsed, and becomes enriched for H3K9me2. (C, D) Examples where full pairing of the intact homologs is disrupted by pairing with sDp3.

chromosomes and chromosomal regions and (ii) targets histone methyltransferase (HMTase) activity to those regions. Presumably the (relatively low level of) HMTase activity is targeted to paired homologs via another mechanism. MET-2, a candidate HMTase, is required for all H3K9me2 marks in the germ line (Bessler et al., 2010). Proteins that methylate histone lysine residues typically contain a SET (Su(var)3–9, Enhancer of zeste, Trithorax) domain, which is required for catalytic activity. met-2 was originally analyzed as a part of a systematic survey of the 38 C. elegans SET domain protein-coding genes and their potential role in somatic (vulval) development (Andersen and Horvitz, 2007). By indirect immunofluorescence, H3K9me2 is not detected in met-2 mutant germ lines, even in mutant backgrounds where ectopic H3K9me2 would normally be present (Bessler et al., 2010; E. Maine, unpublished data). However, germ line H3K9me3 is normal in met-2 mutants (Bessler et al., 2010).

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Interestingly, Andersen and Horvitz (2007) implicated MET-2 in H3K9 trimethylation in the soma. They identified MET-2 as partially redundant with another SET domain protein, MET-1, in vulval development. MET-2 is an ortholog of human SETDB1, an H3K9 methyltransferase; and MET-1 is an ortholog of S. cerevisiae Set2, an H3K36 methyltransferase. Using quantitative protein blots, the authors demonstrated that trimethylation of H3K9 and H3K36 were both reduced in met-1 and met-2 mutant embryos, although H3K9me3 was more severely reduced in met-2 mutants, and H3K36me3 was more severely reduced in met-1 mutants. The authors concluded that MET-2 was primarily responsible for H3K9me3 and MET-1 was primarily responsible for H3K36me3 in the embryo. However, they did not examine H3K9 dimethylation in the met-2 soma, therefore it is not known if MET-2 promotes this mark. Presumably the specificity of MET-2 action, that is, as a di- versus trimethyltransferase, is modulated via interactions with other factors, which may be tissue (e.g., germ line) specific.

3.5. Targeting mechanisms Genetic and molecular studies have identified some of the factors responsible for H3K9me2 (Table 2.2) and H3K27me3 accumulation in the C.elegans germ line. At present, completely independent mechanisms appear to be responsible for targeting these modifications to appropriate sites. One common feature of the two mechanisms is that disruption of each can result in inappropriate deposition of silencing marks on autosomes, as described below. 3.5.1. Regulation of germ line H3K9me2 by siRNA-mediated pathway How is MET-2 activity targeted to unpaired chromosomes during meiosis? One important regulatory mechanism involves small RNAs. The specific accumulation of H3K9me2 on unpaired chromosomes requires activity of a small RNA-mediated pathway whose members include: EGO-1, an RNA-directed RNA polymerase (RdRP); CSR-1, an Argonaute protein; DRH-3, a DEAH/D-box helicase; and EKL-1, a Tudor domain protein (Maine et al., 2005; She et al., 2009). These four proteins function in small RNA-mediated processes such as RNAi and cosuppression (Aoki et al., 2007; Duchaine et al., 2006; Kim et al., 2005; Robert et al., 2005; Smardon et al., 2000; Yigit et al., 2006) and have been shown to interact genetically with components of the Ras/Raf and GLP-1/Notch signaling pathways (Qiao et al., 1995; Rocheleau et al., 2008; She et al., 2009). Recently, they were shown to participate in a biochemical network that produces and utilizes a subclass of small-interfering RNAs (siRNAs) (Claycomb et al., 2009; Gu et al., 2009; van Wolfswinkel et al., 2009). Directly relevant to meiotic silencing, this pathway is required for mitotic chromosome segregation in

Table 2.2 Regulators of H3K9me2 accumulation in the C. elegans germ line Gene

Product

H3K9me2 phenotype

References

met-2 ego-1

Histone methyltransferase RdRP family

Bessler et al. (2010) Maine et al. (2005)

csr-1

Argonaute family

drh-3

DEAH-box helicase

ekl-1

Tudor domains; methyl-binding

chk-2

Kinase

him-17 rha-1 sin-3

Chromatin-binding protein RNA helicase A HDAC complex assembly

H3K9me2 absent No H3K9me2 enrichment on unpaired chromatin in XX or XO germ line H3K9me2 reduced on unpaired chromatin and elevated/ectopic at many paired sites H3K9me2 reduced on unpaired chromatin and elevated/ectopic at many paired sites H3K9me2 reduced on unpaired chromatin and elevated/ectopic at many paired sites No H3K9me2 enrichment on male X; delayed H3K9me2 accumulation in XX germ line Reduced/delayed H3K9me2 accumulation H3K9me2 reduced/absent across genome No H3K9me2 enrichment on unpaired chromatin in XX germ line

She et al. (2009) She et al. (2009) She et al. (2009) Bessler et al. (2007)

Reddy and Villeneuve (2004) Walstrom et al. (2005) She et al. (2009) and X. She and E. Maine (unpublished data)

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the embryo (Claycomb et al., 2009) (see below). All known components of this functional pathway are essential for fertility (Duchaine et al., 2006; Qiao et al., 1995; She et al., 2009; van Wolfswinkel et al., 2009; Yigit et al., 2006). Loss of EGO-1, CSR-1, EKL-1, and DRH-3 activity has drastic effects on H3K9me2 accumulation during meiosis. Interestingly, two different defects are observed. In ego-1 mutants, H3K9me2 does not become enriched on unpaired DNA, and the overall level of germ line H3K9me2 is very low (Maine et al., 2005). EGO-1 activity is not absolutely required for H3K9me2 deposition in germ cells, because the basal level of H3K9me2 normally observed on autosomes is still present in ego-1 mutants (Maine et al., 2005). Loss of H3K9me2 enrichment on unpaired chromosomes was observed in both ego-1 null [ego-1(0)] mutants and in animals carrying one copy of a null mutation and one copy of a point mutation at a conserved residue in the putative RdRP catalytic domain [ego-1(RdRP/null)]. Therefore, RdRP activity seems to be specifically required for H3K9me2 enrichment. Cellular RdRPs are responsible for synthesis of siRNAs from RNA templates (Aoki et al., 2007; Makeyev and Bamford, 2002; Pak and Fire, 2007; Sijen et al., 2001, 2007). An obvious hypothesis is that EGO-1 may be responsible for synthesis of siRNAs that target MET-2 and/or other chromatin modifiers to unpaired DNA. A second H3K9me2 phenotype is observed in csr-1, ekl-1, and drh-3 mutants. In mutant males, the H3K9me2 level is partially reduced on the X chromosome and is elevated on the autosomes (She et al., 2009). Here, unlike in ego-1 mutants, H3K9me2 is inappropriately deposited on paired chromosomes. In this study, She and colleagues identified the X chromosome based on an absence of histone activation marks and a condensed morphology. In contrast to wild-type gonads, when dissected csr-1, drh-3, and ekl-1 gonads were colabeled for H3K9me2 and an activation mark, H3K4me2, the two marks were observed to colocalize at many autosomal sites. This phenotype is consistent with MET-2 HMTase activity being mistargeted in csr-1, ekl-1, and drh-3 mutants and suggests that the activity of CSR-1, EKL-1, and DRH-3 ultimately attracts MET-2 to unpaired chromosomes or excludes MET-2 from paired chromosomes. She and colleagues performed careful analysis of meiotic pairing and synapsis in these mutants to rule out the possibility that pairing or synapsis defects were responsible for the autosomal H3K9me2 foci. All chromosomes appeared to be synapsed based on distribution of HIM-3, an axial component, and SYP-1, an inner component of the synaptonemal complex. When homolog pairing was analyzed using FISH to visualize the 5S ribosomal RNA gene cluster located on LGV, a minor pairing defect was observed in drh-3 and ekl-1 mutants. However, the frequency of nuclei where chromosome V was unpaired was much lower than the frequency of nuclei with ectopic H3K9me2, indicating H3K9me2 was abnormally present at paired chromosomal sites in csr-1, ekl-1, and drh-3 mutants. H3K9me2 accumulation was also abnormal in csr-1, ekl-1, and drh-3 mutant hermaphrodites (XX) germ lines if unpaired chromosomes or a

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chromosomal duplication was present. Normally, H3K9me2 marks are enriched on the chromosomal duplication, sDp3, and on unpaired X chromosomes in him-8 XX mutant hermaphrodites (Bean et al., 2004). This enrichment is reduced or absent in csr-1, ekl-1, and drh-3 XX mutants, and H3K9me2 is elevated on other chromosomes (She et al., 2009). These findings suggest a role for the small RNA machinery in directing MET-2 HMTase activity to unpaired chromosomes and/or away from paired chromosomes. One can ask whether H3K9me2 accumulation is actively targeted to unpaired chromosomes or, in contrast, is simply unable to occur when chromosomes are paired. Ectopic H3K9me2 accumulation on paired chromosomes in certain mutant backgrounds provides evidence for a mechanism to actively prevent H3K9me2 accumulation on those chromosomes. Interestingly, the activity of another RdRP, called RRF-3, is also important for regulation of H3K9me2 levels during meiosis (Maine et al., 2005). Normally, H3K9me2 enrichment disappears from the X chromosome as germ cells undergo spermatogenesis such that very little enrichment is observed in primary spermatocytes. In rrf-3 mutants, in contrast, H3K9me2 foci are clearly visible in primary spermatocytes. Therefore, H3K9me2 appears to turn over more slowly in these mutants, and H3K9me2 levels may be more highly elevated on the X chromosome, as well. RRF-3 activity is known to be required for activity of the so-called ERI (enhanced RNAi) pathway and for biogenesis of certain siRNAs (Gent et al., 2009; Han et al., 2009; Pavelec et al., 2009; Simmer et al., 2002; and references therein). Evidence suggests that EGO-1 and RRF-3 RdRPs have both interrelated and independent functions (see Gent et al., 2010 and references therein). Prolonged H3K9me2 enrichment in rrf-3 mutants may indicate that ERI pathway activity directly limits H3K9me2 enrichment. Alternatively, when RRF-3 is absent, EGO-1 may be more readily available to participate in chromatin regulation. 3.5.2. SiRNA functional pathways in the C. elegans germ line Multiple siRNA-mediated pathways are active in the C. elegans germ line (Claycomb et al., 2009; Gent et al., 2009; Gu et al., 2009; Han et al., 2009; Pak and Fire, 2007; Ruby et al., 2006; van Wolfswinkel et al., 2009). A number of recent studies have combined genetic and deep sequencing approaches to elucidate interrelated small RNA-mediated pathways in the germ line and soma. In the germ line, two classes of 22G-RNAs (22 nucleotide siRNAs containing a 50 guanosine) have been described, both of which required DRH-3, EKL-1, and RdRP activity for synthesis but function in conjunction with different Argonaute proteins (Gu et al., 2009). One class of 22G-RNA corresponds to expressed, protein-coding genes and loads specifically onto CSR-1/Argonaute. EGO-1 is responsible for biogenesis of this class of 22G-RNAs. This functional pathway participates in H3K9me2 regulation during meiosis, presumably by regulating MET-2

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complex activity (Maine et al., 2005; She et al., 2009). Intriguingly, this pathway participates in kinetochore formation and mitotic chromosome segregation in the early embryo, and has been shown to associate with mitotic chromosomes (Claycomb et al., 2009). An obvious hypothesis is that these factors also associate with chromatin in the meiotic germ line. A second class of 22G-RNA produced by EGO-1 and another RdRP, RRF-1, is loaded on WAGO-class Argonautes. This pathway targets transposase transcripts, aberrant transcripts, and pseudogene transcripts, presumably for degradation via a conventional posttranscriptional silencing mechanism. Additional components of these two pathways have been identified, including the b-nucleotidyl transferases CDE-1 (CSR-1 pathway) and RDE-3 (WAGO pathway), and MUT-7/RNaseD (WAGO pathway) (Gu et al., 2009; van Wolfswinkel et al., 2009). Evidence from Schizosaccharomyces pombe suggests that b-nucleotidyl transferase activity determines whether or not an RdRP complex can recognize a particular RNA as substrate (Motamedi et al., 2004). The existence of distinct b-nucleotidyl transferases required for recognition of distinct sets of RNA templates is consistent with a need to recruit different RdRP complexes to different templates. How does this regulatory network influence chromatin regulation? In the absence of EGO-1 activity, very few CSR-1 class 22G-RNAs are produced. In the absence of EKL-1 activity, very few 22G-RNAs of either class are produced. In the absence of DRH-3 activity, fewer 22G-RNAs of both classes are produced and they are biased toward the 30 end of the gene (as if DRH-3 is critical for RdRP processivity). In the absence of CSR-1 activity, 22G-RNAs may be inappropriately loaded onto other Argonautes. It would be useful to know if the 22G-RNA pool is altered in mutants with unpaired chromosomes, for example, him-8 or zim-2 mutants. In other words, does the presence of unpaired chromosomes lead to altered RdRP activity? Also, we do not know if there are differences between the X chromosome-derived 22G-RNAs in XO versus XX animals. Interestingly, many components of the CSR-1 and WAGO siRNA pathways are reported to localize to germ line P granules (Claycomb et al., 2009; Gu et al., 2009), and P granule morphology is altered in germ lines mutant for ego-1, csr-1, drh-3, cde-1, and ekl-1 (Claycomb et al., 2009; Updike and Strome 2009; Vought et al., 2005). An emerging model, based on recent systematic searches for P granule components, is that the P granule serves as a site where the quality of RNAs is assessed as they are exported from the nucleus (Updike and Strome 2009, 2010). 3.5.3. Other germ line regulators of H3K9me2 The function of several other proteins has also been linked to meiotic H3K9me2 accumulation, including RNA helicase A (RHA-1; Walstrom et al., 2005), HIM-17 (a THAP domain chromatin-binding protein; Reddy and Villeneuve, 2004), CHK-2 (related to csd1/chk2 kinases; Bessler et al.,

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2007), and SIN-3 (a putative component of type III histone deacetylase (HDAC) complexes; She et al., 2009; X. She and E. Maine, unpublished data). Reduction or loss of function of each of these proteins changes the distribution of H3K9me2 in the meiotic germ line. Loss of RHA-1 function severely reduces H3K9me2 accumulation on transgenic arrays and the male X chromosome in meiotic germ cells in mutants raised at high temperatures (Walstrom et al., 2005; X. She and E. Maine, unpublished data). In addition, activation marks (e.g., H3K4me2 and H4K16ac/H4K8ac) appear on all chromosomes, including the condensed X chromosome (Walstrom et al., 2005). Some aspects of the rha-1 (null) phenotype are temperature sensitive: germ line development and meiotic H3K9me2 accumulation are disrupted in rha-1 mutants at restrictive temperature but not at permissive temperatures. The rha-1(0) mutant also is partially defective in germ line RNAi, a phenotype that is not subject to temperature. One model is that RHA-1 is partially redundant for function with another RNA helicase capable of substituting for RHA-1 at lower temperatures but incapable of doing so at higher temperatures. HIM-17 is a chromatin-binding protein implicated in H3K9me2 enrichment in both the XX and XO germ line. H3K9me2 enrichment on the male X and at localized regions in the XX genome is reduced and delayed in him-17(null) mutants (Reddy and Villeneuve, 2004). In contrast, the basal level of H3K9me2 present across the genome seems to be normal. Genetic analysis indicated that HIM-17 is required for double-strand break formation but not for synapsis. These findings suggest a link between chromatin regulation and double-strand break formation. CHK-2, a member of the checkpoint kinase family, is required for cell cycle arrest and apoptosis in the germ line in response to UV-induced DNA damage (Stergiou et al., 2007). CHK-2 activity is also required for pairing (MacQueen and Villeneuve, 2001) and, consistent with this observation, CHK-2 has been shown to phosphorylate several target proteins during leptotene/zygotene stage of meiotic prophase I (Penkner et al., 2009). H3K9me2 accumulation is severely reduced in chk-2 males and delayed in chk-2 hermaphrodites (Bessler et al., 2007). Perhaps one or more targets of CHK-2 kinase activity promote meiotic H3K9me2 accumulation. SIN-3 is the sole C. elegans ortholog of mammalian Sin3A and SIN3B proteins. Various studies in mammals and yeast have shown that Sin3 proteins bind HDACs as well as proteins involved in nucleosome remodeling, DNA methylation, N-acetyl-glucosamine transferase activity, histone methylation, and transcriptional control (Cunliffe, 2008; Silverstein and Ekwall, 2005). Evidence suggests that C. elegans SIN-3 activity may be particularly critical for meiotic silencing of chromosomes other than the male X (X. She and E. Maine, unpublished data). One model is that SIN-3 deacetylase activity may allow or promote H3K9 methylation. An

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alternative model is that SIN-3 recruits chromatin or nucleosome remodelers whose activity promotes MET-2 activity.

3.6. Enrichment of H3K27me3 on X chromosomes The accumulation of H3K9me2 on unpaired chromosomes during early meiotic prophase is superimposed on other chromatin marks that may be present in the mitotic germ line. As described above, there is a general lack of histone activation marks on X chromosomes in mitotic nuclei in both the XX and XO germ line. In addition, the X chromosome is enriched for H3K27me3, a histone modification strongly associated with transcriptional repression. This general X-specific enrichment for H3K27me3 was discovered by Strome and colleagues as a consequence of their analysis of the maternal effect controls on germ line development. Capowski et al. (1991) isolated mutations in a set of mes (maternal-effect sterile) genes whose expression in the maternal germ line promotes survival and development of germ cells in their progeny. Four genes, mes-2, -3, -4, and -6, promote germ cell survival in the larva; in the absence of MES-2, -3, -4, and -6 function, germ cells tended to degenerate during larval development and rarely produced gametes (Capowski et al., 1991; Garvin et al., 1998; Paulsen et al., 1995). The initial indication that MES protein activity might regulate X chromosome function in the germ line was provided by genetic analysis of mes phenotypes in XO, XX, and XXX animals (Garvin et al., 1998). The severity of the mes-2, -3, -4, and -6 mutant phenotypes was strongly influenced by X chromosome dose; among progeny of mes mutant mothers, XO animals had (on average) the mildest phenotype while XXX animals had the more severe phenotype. Using sex determination mutants, Garvin et al. (1998) demonstrated that germ cell survival was linked to X chromosome number rather than sexual identity. One interpretation of these data was that elevated X-linked gene expression in mes mutants caused germ cell degeneration; the greater the number of X chromosomes, the stronger the degenerative phenotype. Overall, genetic data were consistent with the hypothesis that MES-2, -3, -4, and -6 function in a mechanism to limit the expression of X-linked genes in the germ line. Molecular studies revealed a role for the MES system in regulating X chromatin in the germ line. Initially, two different lines of evidence pointed in this direction. MES-2 and MES-6 proteins were shown to be members of the Polycomb group (PcG) family (Holdeman et al., 1998; Korf et al., 1998), proteins later shown to have histone-modifying activity. At the same time, Kelly and Fire (1998) identified mes-2, -3, -4, and -6 as regulators of transgene silencing: an extrachromosomal transgenic array, normally silenced in the germ line, was expressed (‘‘desilenced’’) in mes mutant germ lines. Indirect immunofluorescence analysis indicated that H3K9me2

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distribution was normal in the mes mutants; hence, the MES system appeared to regulate transgenes independently of the meiotic silencing process (Fong et al., 2002). Later studies showed that MES-2, -3, and -6 promote histone H3 lysine 27 di- and trimethylation in the germ line (Bender et al., 2006). In wild type, these two marks are present on all germ line chromosomes, but H3K27me3 is particularly enriched on X chromosomes (Bender et al., 2006). In addition, MES-2, -3, and -6 function to limit the accumulation of histone activation marks on the X chromosome (Fong et al., 2002).

3.7. Regulation of germ line H3K27me3 by Polycomb Repressive Complex 2 (PRC2) system Three of the four MES proteins, MES-2, -3, and -6, appear to function in a protein complex required for H3K27 methylation in the germ line. MES2 and MES-6 are orthologs of PcG proteins Enhancer of zeste [E(z)] and extra sex combs, respectively (Holdeman et al., 1998; Korf et al., 1998). MES-3 is a novel protein found in complex with MES-2 and MES-6 (Paulsen et al., 1995; Xu et al., 2001a,b). MES-2 has H3K27 methyltransferase activity, and MES-6 and MES-3 are required for this activity in vitro (Ketel et al., 2005). Consistent with the hypothesis that MES-2/-3/-6 complex is responsible for depositing H3K27me3 onto the X chromosome, these proteins are detected in the nucleoplasm (Holdeman et al., 1998; Korf et al., 1998; Xu et al., 2001a). They presumably act directly on chromatin, although a direct association has not been observed. In contrast to other identified components of the MES system, MES-4 has H3K36 methyltransferase activity and does not physically interact with MES-2, -3, or -6 (Bender et al., 2006; Xu et al., 2001a). MES-4 localizes to the autosomes and active transgene arrays and is largely absent from X chromosomes and silenced transgene arrays (Fong et al., 2002). In the embryo, activity of the MES-2/-3/-6 complex prevents MES-4 from associating with the (oocyte derived) X chromosome (Fong et al., 2002), suggesting that inappropriate MES-4 activity on the X may cause the germ line defects observed in mes-2, -3, and -6 mutants. Interestingly, MES-4 is never observed to associate with the sperm-derived X, perhaps due to the imprint described by Bean et al. (2004) (discussed in Section 4.3). Data suggest that MES-4 is not simply a positive regulator of gene expression. Typically, H3K36me3 marks are found within the exons of active genes (Kolasinska-Zwierz et al., 2009). However, MES-4 does not colocalize with activated RNA pol II, and microarray analysis of mes-4 mutants revealed little change in autosomal gene expression; instead, a subset of X-linked genes was upregulated, suggesting that the primary function of MES-4 activity on the autosomes is to limit X chromosome gene expression (Bender et al., 2006). Alternatively, MES-4 activity on autosomes may limit MES-2/-3/-6 complex activity to the X, ensuring

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H3K27me3 enrichment on the X. Another function for MES-4 may be to mark genes that should be activated in the germ line. In a mutant where MES-4 abnormally associates with the X, some X-linked genes would be inappropriately marked and therefore expressed, in turn impairing germ line function. In addition to the known MES proteins, other regulators are clearly involved in X chromosomal repression because the pattern of H3K27me3 enrichment across the genome is grossly normal in the germ line of mes-4 mutants. For example, the absence of MES-4 activity does not lead to elevated H3K27me3 on autosomes or to a visible loss of H3K27me3 on the X chromosome (Bender et al., 2004). The mechanisms targeting MES2/-3/-6 and MES-4 are unclear, although one obvious model is that noncoding RNA (ncRNA) may participate in the targeting process given the recent data demonstrating interactions between ncRNAs and PRC2 in mammalian tissues (discussed in Section 6, below). Another regulator of X chromosome silencing, the chromodomain protein MRG-1, shares many features in common with MES-4. MRG-1, like MES-4, ensures survival of primordial germ cells, promotes silencing of X-linked genes in the XX germ line, promotes silencing of extrachromosomal arrays in the (XX and XO) germ line, and localizes to autosomes (Fujita et al., 2002; Takasaki et al., 2007). Similarly, the mrg-1 phenotype is much less severe in males than in hermaphrodites. The relationship between MRG-1 and MES-4 is unclear at present, although MRG-1 can associate with autosomes even in the absence of MES-4. Other regulators of X chromosome silencing in the germ line include histone-modifying enzymes and histone variants. For example, the histone variant, HIS-24/H1.1, and SIR-2.1 deacetylase appear to promote H3K27me3 deposition in the germ line ( Jedrusik and Schulze, 2007; Wirth et al., 2009). Based on indirect immunofluorescence analysis of mutants, the loss of SIR-2.1 activity correlates with increased H3 acetylation, and methylated H3 lysine 9 does not appear on repetitive transgene arrays in his-24 mutants. These findings suggest a link between H3K9 deacetylation, H3K9 and H3K27 methylation, and HIS-24 activity. Analysis of the H3K9me2 distribution in his-24 males has not been reported. It would clearly be of interest to determine whether HIS-24 activity is linked to H3K9me2 enrichment on unpaired chromosomes. As another example, mes-3 and mes-4 phenotypes are enhanced by the loss of SET-2 function. SET-2 is a putative H3K4 methyltransferase related to mammalian SET1/MLL (Simonet et al., 2007; Xu and Strome, 2001). Presumably inappropriate H3K4 methylation of active genes on the X chromosome in mes-3 and mes-4 mutants contributes to their phenotype. Therefore, it is not immediately clear why the loss of H3K4 methylation exacerbates the mes phenotype. Perhaps loss of SET-2 function causes a widespread misregulation of germ line gene expression that exacerbates the ill health of mes-3 and mes-4 mutant germ cells.

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3.8. Summary In summary, two distinct mechanisms are known to regulate the X chromosome in the germ line. (i) The meiotic silencing system is active during early meiotic prophase, and targets the H3K9me2 modification to the single (male) X chromosome. In mutant backgrounds where other chromosomes are unpaired/unsynapsed, the meiotic silencing system targets H3K9me2 to those chromosomes. (ii) The MES system is active in mitotic and meiotic germ cells and ensures survival/function of the germ line. MES activity is responsible for histone modification across the genome, although the X appears to be a preferential target. H3K27me3 is well documented to correlate with transcriptional quiescence in a variety of organisms, whereas the link between H3K9me2 and transcriptional repression is less firm. In the C. elegans germ line, chromosomal regions can receive H3K9me2 yet remain active, raising interesting questions as to the function of this modification. Perhaps H3K9me2 has a different effect in different chromatin contexts, for example, when accumulating on chromosomal regions entering meiosis in an active state (autosomes) or in a silent state (X chromosomes). Although progress has been made toward understanding the mechanisms for targeting H3K27me3 and H3K9me2 to appropriate sites, many questions still remain. Clearly, identification of the relevant histone modifiers is an important step. The next step is to understand how these modifiers are directed to appropriate chromosomal locations. Although the mechanisms described above are distinct, one intriguing parallel is both mechanisms appear to maintain a balance between silencing of the correct targets and incorrect targets. For example, CSR-1, EKL-1, or DRH-3 activity allows MET-2, preferentially active on the male X, to have elevated activity on paired/synapsed autosomes. Similarly, loss of MES-2/-3/-6 activity allows MES-4, normally active on autosomes, to associate with the X.

4. Meiotic Silencing and Germ line Development in C. elegans The function of H3K9me2 enrichment is unclear, however it does not appear simply to correlate with the repression of gene expression. Unpaired homologs remain transcriptionally active as evidenced by the presence of histone activation marks (Bean et al., 2004; Jaramillo-Lambert and Engebrecht, 2010) and by extensive genetic data. Mutations such as him8 and zim-2 do not cause major germ line developmental defects; hence, expression of essential maternal effect genes in the germ line is not grossly abnormal in these mutants (Brenner et al., 1979; Phillips and Dernburg,

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2006; see other examples discussed below). Moreover, free chromosomal duplications are routinely used in as balancers and in gene dosage studies to demonstrate that a mutation with a germ line phenotype is strictly recessive loss of function. For example, the recessive sterile phenotype of glp-1 mutants (glp-1(/)) is rescued in glp-1(/); mnDp37 animals by expression of the wild-type copy of glp-1 present on mnDp37 (Austin and Kimble, 1987). Such data suggest that chromosomal regions containing expressed genes can also be enriched for H3K9me2, consistent with the hypothesis that H3K9me2 may accumulate on unpaired chromosomes for a purpose other than—or in addition to—silencing transcription. One caveat is the likelihood that the duplication is paired with an intact homolog in some germ line nuclei (as depicted in Fig. 2.3), and expression of the gene in question in those nuclei may be sufficient to rescue the mutant phenotype. Relevant here is the observed repression of X-linked genes in XO oogenesis (Bean et al., 2004; Jaramillo-Lambert and Engebrecht, 2010; see Section 4.4), which seems to indicate that the regulation of the single X chromosome is different from that of other unpaired chromosomes. Perhaps the meiotic silencing system cannot shut down expression of genes that enter meiosis in an active state (e.g., autosomal genes) but is able to maintain repression of the X, which is silent prior to pachytene. There is considerable speculation in the literature as to the function(s) of meiotic silencing in animals species. In C. elegans, the situation is obviously complicated by the presence of the MES system, a general repressive mechanism that appears to be responsible for repression of X-linked gene expression per se. Moreover, the appearance of H3K9me2 on unpaired homologs does not correlate with wholesale reduction in transcription of genes on those chromosomes. In the male, where MES activity does not seem to be essential for germ line survival, H3K9me2 accumulation on the X may provide partial functional redundancy in silencing gene expression, allowing germ line survival and function. However, it seems very likely that H3K9me2 enrichment on unpaired chromosomes, per se, has a different function. Part of the difficulty in understanding the importance of regulating unpaired chromatin is that we do not know the biological consequences of the H3K9me2 mark (or most other chromatin marks, for that matter). Certain modifications correlate with increased or decreased transcription, but cause and effect is rarely clear (e.g., see Wu et al., 2009). During C. elegans meiosis, chromatin structure may be controlled for other purposes besides or in addition to transcription, that is, to prevent unpaired chromatin from tripping a checkpoint, for example, or to allow chromosomes to progress through pachytene. Another observation to bear in mind is that the loss of H3K9me2 accumulation on the X chromosome in ego-1 mutant males did not prevent the chromosome from undergoing its typical condensation or allow activation marks to accumulate (Maine et al., 2005). Hence, the male X is subject to more than one regulatory process. Perhaps

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H3K9me2 deposition on the male X, made within the context of other regulatory mechanisms active on that chromosome, has a different outcome than it does within the context of actively expressed autosomal and hermaphrodite X chromosomes. The various proposed functions of meiotic silencing in animals are discussed below in terms of their applicability to C. elegans. In addition to these considerations, H3K9me2 accumulation is not strictly required for production of normal sperm, because met-2 mutant males are fertile (Bessler et al., 2010). It is not yet clear if these mutants have reduced fertility, particularly over time. met-2 mutant hermaphrodites are reported as having a mortal germ line (Mrt) phenotype whereby homozygous mutants become sterile after 18–24 generations (Andersen and Horvitz, 2007). The Mrt phenotype is more severe in met-1; met-2 double mutants, suggesting that the simultaneous loss of multiple histone marks leads to gradual loss of germ line viability and function. It seems surprising that the complete loss of a conserved histone modification would have little apparent effect on tissue function. One explanation for the relatively mild met-2 phenotype might be that H3K9me2 marks are partially redundant with other histone marks. Alternatively, H3K9me2 accumulation might be more critical in certain environmental conditions not commonly found in the laboratory.

4.1. Repression of DNA insertions Meiotic silencing in N. crassa represses expression of transposons and other DNA insertions. Might C. elegans meiotic silencing have a similar function? For example, transcription of DNA insertions might be reduced as a consequence of H3K9me2 accumulation. This function would limit both the detrimental expression of foreign DNA during gametogenesis and the mutagenic effect of transposons. However, transposon activity is repressed via a posttranscriptional mechanism active throughout the germ line (Girard and Hannon, 2008; Golden et al., 2008; Sijen and Plasterk, 2003), which might obviate any need for chromatin-based repression that the meiotic silencing system could provide. Furthermore, many questions remain about how chromatin-based transposon silencing would work in C. elegans. As discussed above, unpaired chromosomal duplications are not transcriptionally silenced despite the presence of H3K9me2 marks. Hence, there is no evidence that an unpaired transposon or DNA insertion would be transcriptionally silenced. On a related point, nothing is known about the lower size limit of the H3K9me2 enrichment phenomenon, that is, we do not know how large an unpaired region must be in order to receive the mark. It is not clear whether a single unpaired transposable element could be detected and targeted for H3K9me2 accumulation. These questions will be addressed by the identification of specific sites that become enriched for H3K9me2 during meiotic prophase.

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4.2. Maintenance of genome integrity Another hypothesis is that meiotic silencing mechanisms function in maintaining genome integrity during gametogenesis. The stability and segregation of unpaired chromosomes during meiotic prophase and the meiotic divisions, respectively, may require establishment of particular chromatin structure. A related idea is that meiotic silencing may be important for checkpoint control. There is a meiotic checkpoint in place to detect delays in pairing/synapsis, however in XO individuals of either sex the X chromosome is not subject to this control ( Jaramillo-Lambert and Engebrecht, 2010). In other words, the single X chromosome somehow escapes detection by the checkpoint system. One intriguing idea is that the specialized chromatin structure of the single X shields it from detection as unsynapsed (see Gartenberg, 2009; Handel 2004; Kelly and Aramayo 2007). Such functions would be critical for completion of meiosis and, consequently, fertility. The effect may be minor in most nuclei, but the cumulative effect over generations might be large. For example, met-2 mutant hermaphrodites (which lack germ line H3K9me2) exhibit a variety of germ line defects, each only weakly penetrant (Bessler et al., 2010) but which may contribute to the mortal germ line phenotype observed over many generations (Andersen and Horvitz 2007).

4.3. Epigenetic control of embryogenesis Meiotic silencing may contribute to the establishment of heritable chromatin marks during spermatogenesis that persist in the early embryo (imprinting). In many organisms, differences between activation of maternally versus paternally inherited genes have been observed. Such differences in gene expression are thought to reflect the patterns of histone marks inherited from each parent, for example, the paternal versus maternal X chromosomes may contain different patterns of histone marks. Such marks might promote embryonic viability and development, including eventual fertility of the offspring (Bean et al., 2004; Huynh and Lee, 2003; Strome, 2005; Turner, 2007; Turner et al., 2006). Bean et al. (2004) compared the chromatin state of the maternal and paternal X chromosomes in XX C. elegans embryos. They observed differential regulation of a single chromosome from the sperm pronucleus, which failed to label with antibodies against the activation marks H3K4me2 and H3K9/K14ac. Most sperm-derived chromosomes accumulated these activation marks prior to the first cell division; however, these activation marks did not appear on the sperm-derived X chromosome until the embryo had undergone several rounds of cell division. Interestingly, late activation of the sperm X chromosome was observed regardless of whether the sperm was derived from an XO male or an XX hermaphrodite, suggesting an X chromatin imprint is

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characteristic of sperm per se. However, the parental origin of the X chromosome did seem to influence the timing of imprint loss: H3K4me2 marks were detected on the hermaphrodite sperm-derived X by 10–14 cell stage, whereas they were not visible on the male sperm-derived X until the 12–20 cell stage. Bean and colleagues hypothesize that the late loss of the X chromatin imprint is related to the unpaired status of the male-derived X chromosome. Consistent with this model, the X chromatin imprint is lost early in the progeny of XX tra-2 males—where the X chromosomes are paired throughout meiosis as they are in normal hermaphrodites. Recently, Hammoud et al. (2009) showed that chromatin marks present in human sperm are transmitted to offspring. These marks include activation (H3K4me2, H3K4me3) and silencing (H3K27me3) modifications enriched at developmentally important loci. Similarly, there is also some evidence that histone activation marks established in the C. elegans germ line persist in sperm and are transmitted to the embryo ( J. Arico and W.G. Kelly, personal communication). Hence, the transcriptional history of genes in the adult germ line, as reflected in the chromatin state of gametes, may have a direct impact on the epigenetic information inherited by the embryo. We do not yet know the physiological consequences of this differential regulation of maternal versus paternal X chromosomes in the C. elegans embryo.

4.4. Transcriptional regulation of single X Does the meiotic silencing system function in transcriptional repression in the XO nucleus? One way to address this question is to examine the effect of H3K9me2 accumulation on oogenic gene expression in XO hermaphrodites. Mutations in the her-1 gene transform XO soma and germ line to hermaphrodite development (Hodgkin, 1980). In her-1 XO loss-offunction mutants, oocyte production is reduced and average self-progeny viability is only 1% of wild type (Hodgkin, 1980, 1983). Embryonic lethality can be partially explained by the observation that  25–30% of the progeny of XO her-1 hermaphrodites are nullo-X due to loss of the single X chromosome at meiosis (Hodgkin, 1983). However, additional factors must further reduce progeny viability and oocyte production. Bean et al. (2004) analyzed H3K9me2 accumulation in XO null mutants and found that the single X was enriched for H3K9me2 in pachytene–diplotene stages, that is, at the time when there is normally a burst of X-linked transcription. Moreover, the levels of at least some X-linked transcripts are lower in XO versus XX hermaphrodite germ lines as analyzed by in situ hybridization, suggesting that the elevated H3K9me2 may limit transcription. XO her-1(null) hermaphrodites produced oocytes but no viable progeny, leading Bean and colleagues to hypothesize that inappropriate silencing of the single X chromosome during meiosis had impaired oogenesis and resulted in embryonic lethality. It is not clear if the difference

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in brood sizes between the different her-1 alleles is important, however the data are consistent with the hypothesis that H3K9me2 accumulation on the single X may reduce gene expression. In their analysis of checkpoint controls, Jaramillo-Lambert and Engebrecht (2010) showed that transcription from the single X chromosome in XO fem-3 hermaphrodites is also inappropriately silenced during late pachytene. Hence, the single X chromosome is subject to unique regulation regardless of germ line sex.

4.5. Chromosome evolution Over time, differential regulation of the heterogametic sex chromosomes may also have influenced chromosome evolution. In nematodes, X-linked essential genes often have an autosomal paralog that is active throughout germ line development (Maciejowski et al., 2005; Ohmachi et al., 2002). In males, in particular, very few X-linked genes are expressed in the germ line. The dearth of X-linked gene expression in the germ line may be an adaptation to prevent silencing of essential genes. Meiotic silencing may aid in repressing/ensuring the low level of X-linked gene expression; as described above, in situ hybridization experiments indicate that X-linked oogenic genes fail to express properly in the feminized XO germ line, an outcome that suggests the single X chromosome is transcriptionally silenced (Bean et al., 2004; Jaramillo-Lambert and Engebrecht, 2010). It is difficult to know which came first, migration of germ line-essential genes off of the X or establishment of repressive X chromatin structure.

4.6. Summary Hypotheses abound as to the possible function(s) of meiotic silencing. To resolve this question for C. elegans, we need to identify the targets of H3K9me2 modification and better understand the effect of such modifications on chromatin structure. Another consideration to bear in mind is the possibility that meiotic H3K9me2 enrichment on the single X chromosome in the XO germ line may produce a different—or partially overlapping—set of outcomes than does H3K9me2 enrichment on homologs (autosomes or Xs) that remain unpaired/unsynapsed due to mutation.

5. Mechanistic and Functional Comparison of Meiotic Silencing Phenomena in Different Species As discussed in Section 1, the term meiotic silencing is applied to a variety of phenomena in diverse species. As these processes have been studied, researchers have uncovered similarities and differences among them with

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regard to the mechanisms involved and effects on gene expression. Many mechanistic details of meiotic silencing seem to be integrated with other, simultaneous processes occurring in the germ line, as described below.

5.1. Meiotic silencing in N. crassa Meiotic silencing is best understood in the fungus, N. crassa, where it targets expression of individual unpaired genes and paired genes whose homology to each other is lower than a particular threshold. In marked contrast to C. elegans and other animal models, evidence suggests that meiotic silencing in N. crassa occurs at a posttranscriptional level. For example, meiotic silencing elicited by a chromosomal deletion will target transcripts produced from paired copies of the deleted region (present as transgenes) that are located at a distinct site (Lee et al., 2004; Shiu et al., 2001). Similarly, the presence of a chromosomal duplication will trigger silencing of transcripts produced from the corresponding paired regions. Components of the silencing pathway also are required for successful meiosis and gametogenesis, perhaps because they function in developmental gene regulation (Shiu and Metzenberg, 2002; Shiu et al., 2001). Meiotic defects may reflect a failure to silence unpaired regions and/or a failure of the MSUD machinery to engage in other regulatory activities. For example, one component is an RdRP whose activity may promote the biogenesis of siRNAs required for developmental regulation of gene expression. Similarly, C. elegans meiotic silencing mutants also tend to be sterile or subfertile. There are striking parallels between meiotic silencing in N. crassa and posttranscriptional mechanisms of transposable element silencing that have been described in C. elegans and other animal species (see Girard and Hannon, 2008). These mechanisms utilize the core RNAi machinery. For example, meiotic silencing in N. crassa utilizes an RdRP (SAD-1), an Argonaute protein (SMS-2), and a Dicer-like protein (SMS-3) (Alexander et al., 2008; Lee et al., 2003; Shiu et al., 2001). Indeed, the most obvious difference described so far between small RNA-mediated transposable element silencing in animal germ lines and MSUC in N. crassa is simply the timing of onset: transposition in animals is repressed throughout the proliferating and meiotic germ line, whereas MSUD specifically initiates in early meiotic prophase. The mechanistic similarities may reflect a common origin for these posttranscriptional silencing processes as adaptations of an ancestral genome defense mechanism.

5.2. Meiotic silencing in insects It has been unclear whether sex chromosomes are silenced during insect meiosis. In Drosophila, X-linked gene expression is similar in male and female germ lines (Gupta et al., 2006). Moreover, synapsis—which

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commonly prevents meiotic silencing—does not occur in the male germ line (although homologs do align) (see Zickler, 2006). Hence, the current thinking is that sex chromosomes are not silenced during meiosis in Drosophila males. In contrast, recent analysis of X chromosome regulation in grasshopper has revealed some features in common with XY-body regulation in mouse (Cabrero et al., 2007). Like C. elegans, the grasshopper Eyprepocnemis plorans lacks a Y chromosome; males are XO. In E. plorans, histone activation marks (e.g., acetylation of H3K9) are observed on the male X chromosome in mitotic germ cells, suggesting some X-linked genes are expressed prior to meiotic entry. These activation marks are lost at the very onset of meiotic prophase (early leptotene stage). By late zygotene stage, the male X chromosome accumulates histone silencing marks and histone variants similar to those associated with MSCI in mouse, including H2AX phosphorylation (gamma-H2AX) (Cabrero et al., 2007). Based on these results, meiotic silencing (or a related process) does appear to occur in E. plorans. Many questions remain to be addressed, including the identity of the silenced genes and whether the silencing mechanism also targets unpaired autosomes or autosomal duplications.

5.3. Meiotic silencing in mammals Chromatin-based silencing of the male sex chromosomes has been described in both eutherians such as mouse (Turner, 2007) and marsupials such as opossum (Franco et al., 2007; Namekawa et al., 2007). The process is best characterized in mouse, where several histone marks accumulate on the XY-body, including H3K9me2 (Khalil et al., 2004), H2A ubiquitination (Baarends et al., 2005; de Vries et al., 2005), and phosphorylation of the histone 2A variant, H2AX (Handel 2004). In addition, some marks of active chromatin are absent from the male sex chromosomes (e.g., H3K9ac, Khalil et al 2004; Namekawa et al., 2006), while other marks are present (e.g., H3K4me2; Khalil et al., 2004). Furthermore, some histones are replaced by variants; for example, H3.1 and H3.2 are replaced with H3.3 (van der Heijden et al., 2007). Interestingly, C. elegans has the reverse situation wherein the single X remains devoid of histone variant H3.3 while paired/synapsed chromosomes accumulate it (Ooi et al., 2006). Disruption of the meiotic silencing process in both mice and opossum leads to elevated expression of X-linked genes as assayed by real-time PCR and by association of active RNA pol II with the sex chromosomes (see Turner, 2007; Zamudio et al., 2008). In normal development, expression of some Y- and Xlinked genes decreases as early as the onset of first meiotic prophase (Wang et al., 2005). Reminiscent of the situation in C. elegans, many X-linked essential genes have an autosomal paralog that is active during male meiosis (Wang, 2004). In mouse, disruptions in male sex chromosome regulation

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correlate with meiotic progression defects and sterility (Handel, 2004; Mahadevaiah et al., 2008; Turner, 2007; Zamudio et al., 2008). As in C. elegans, meiotic silencing in mammals naturally targets the male sex chromosomes, but also targets large unpaired chromosomal regions such as chromosomal translocations that may be present in either the XX or XY germ line. Analysis of mouse strains carrying chromosomal rearrangements or synapsis mutants has shown that unsynapsed autosomes and autosomal translocations are targeted for H3K9me2 and other diagnostic silencing marks during meiotic prophase in the male and female germ line (Baarends et al., 2005; Turner et al., 2005). Histone replacement occurs on both the XY-body and asynapsed autosomes (van der Heijden et al., 2007). Based on the presence of histone variants associated with transcriptional repression and the absence of RNA pol II (as assayed by indirect immunofluorescence), these unsynapsed autosomal regions appear to be silenced. Therefore, a general MSUC phenomenon was judged to be present in mouse, and MSCI is considered to be the ‘‘natural’’ result of MSUC. Recent studies indicate that the meiotic silencing machinery can be overloaded by the presence of multiple unsynapsed chromosomes or translocations, leading to reduced efficiency in silencing the XY-body (Homolka et al., 2007; Kouznetsova et al., 2009; Mahadevaiah et al., 2008). In mouse, defective autosomal synapsis is associated with meiotic arrest and infertility, perhaps due at least in part to a failure to repair double strand breaks (Turner, 2007). The presence of unpaired chromosomal duplications is linked to male meiotic prophase arrest, and defects in pairing or synapsis generally result in inappropriate silencing of the affected regions, which presumably accounts for the subsequent meiotic failure and/or germ cell death (Turner, 2007). It is unclear whether silencing of autosomal genes also contributes to the observed meiotic defects. Clearly, mutations in the MSCI machinery lead to meiotic arrest, however cause and effect is not clear (see discussion in Turner, 2007). It has been suggested that meiotic silencing in this context may function to abort production of sperm with DNA insertions. Alternatively, meiosis may fail because the meiotic silencing machinery may be diverted from regulation of the sex chromosomes to regulation of other unpaired regions (Homolka et al., 2007; Schimenti, 2005; see Turner, 2007). This characteristic of meiotic silencing is distinct from the situation in C. elegans, where unpaired/unsynapsed regions (with the exception of the single X) do not appear to be transcriptionally silenced. The mechanism of meiotic silencing in mouse requires activity of a set of DNA-damage recognition and repair proteins (Turner, 2007 and references therein). In addition, the XY-body accumulates H2A variants associated with the DNA damage response (Turner, 2007). Meiotic silencing is thought to be triggered by the association of BRCA1 protein with unsynapsed chromosomes, in turn recruiting the ATR checkpoint kinase, which

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then phosphorylates H2AX (Fernandez-Capetillo et al., 2003; Turner et al., 2004, 2005). Other unsynapsed regions that may be present (e.g., translocations) also accumulate these factors (van der Heijden et al., 2007). This aspect of the meiotic silencing machinery is apparently not shared with C. elegans because orthologs of many XY-body-associated proteins and histone variants are either absent from C. elegans (e.g., H2AX; http:// www.wormbase.org) or have no apparent role in meiotic silencing (e.g., BRCA1; W.G. Kelly, personal communication).

5.4. Meiotic silencing in birds In chicken, where females are the heterogametic sex, the single Z and W chromosomes are subject to MSCI (Schoenmakers et al., 2009). The observation of MSCI in birds is important because it indicates that this phenomenon is not specific to spermatogenesis, but also functions in oogenesis. As in XX/XY species, meiotic silencing in chicken occurs during pachytene– diplotene stages of first meiotic prophase. The major evidence for MSCI at present is the absence of activated RNA pol II from the ZW chromosome pair and a reduction in Z-linked mRNA transcript levels during meiotic prophase (Schoenmakers et al., 2009). In addition, the ZW pair becomes enriched for H3K9me3 and other histone silencing marks, as well as specialized histone variants typically associated with nontranscribed loci. Very recently, it was shown that a specialized chromosome present in a single copy in male zebra finch (the ‘‘germ line restricted chromosome’’) is silenced during male (ZZ) meiosis, indicating the presence of a general mechanism for silencing unsynapsed chromatin (Schoenmakers et al., 2010). The processes of MSCI in chicken and mouse show several differences with respect to the relative timing of events, for example, the meiotic stage at which gamma-H2AX associates with each sex chromosome. Moreover, the mechanisms of MSCI are different in terms of the importance of the synaptonemal complex. In chicken, Z and W are fully synapsed during meiosis, hence MSCI occurs despite the presence of a synaptonemal complex. This situation is distinct from that in the mouse where failure to synapse is thought to be the trigger for MSCI (see Turner, 2007). Perhaps the failure of homologs to pair (or subsequent nonhomologous synapsis) is the trigger for MSCI in birds. Given these results, different species appear to rely on synapsisdependent or -independent sensing/triggering mechanisms.

5.5. Other meiotic transsensing phenomena in vertebrates Small RNAs have not yet been linked to meiotic silencing in vertebrates, but they have been implicated in other meiotic transsensing phenomena. For example, the mouse MAELSTROM protein is known to physically interact with two Argonaute proteins and is required for transposon repression and

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fertility (Soper et al., 2008). Although initially reported to localize to the XYbody (Costa et al., 2006), subsequent analysis indicated that it is not required for meiotic silencing and does not localize to the XY-body (Soper et al., 2008). More importantly, an epigenetic silencing phenomenon related to paramutation in plants has been reported in mouse (Grandjean et al., 2009; Herman et al., 2003; Rassoulzadegan et al., 2002, 2006; Worch et al., 2008). Paramutation is a phenomenon whereby one allele induces a heritable change in another naive allele present in trans (Chandler et al., 2000). Most of the documented cases involve repression of the naive allele by a ‘‘paramutator’’ allele; the induced allele remains silent even in individuals who do not inherit the original paramutator allele (Chandler et al., 2000). Paramutation does not involve a change in DNA sequence and is presumed to be an epigenetic phenomenon. In plants, paramutation has been shown to require RdRP activity, indicating a probable role for small RNA in establishing the heritable change in gene expression (Alleman et al., 2006, Sidorenko and Chandler, 2008). It is early days for the analysis of paramutation-related phenomena in mammals, however data suggest that small RNA participates in the process (Cuzin et al., 2008). Hence, epigenetic regulation during mammalian meiosis may involve small RNA.

5.6. Summary Based on current data, the importance of meiotic silencing to germ line function in C. elegans is unclear. While many intriguing hypotheses can be proposed, a clear understanding of the biological consequences of H3K9me2 accumulation on unpaired chromosomes awaits further molecular analysis.

6. Noncoding RNA and Chromatin Structure Although the small RNA machinery was first described as regulating gene expression at a posttranscriptional level, these pathways have now been implicated as regulators of chromatin structure, chromosome segregation, and chromosome stability in plants, animals, fungi, and ciliated protozoa (Moazed, 2009; Peters and Meister, 2007; Zaratiegui et al., 2007). Small RNAs participate in posttranscriptional silencing by acting as guides to target an Argonaute protein-containing complex to specific mRNA targets (Hammond, 2005; Peters and Meister, 2007). siRNAs are thought to serve a similar guide function during transcriptional control where they participate in a transcriptional silencing complex. The best-studied example of siRNA-mediated chromatin regulation is the transcriptional silencing of centromeric repeats in S. pombe, which utilizes components of the RNAi machinery to target H3K9me2 marks

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(Djupedal and Ekwall, 2009; Verdel et al., 2009; Volpe et al., 2002; White and Allshire, 2008). This machinery shares many components with the meiotic silencing machinery described in C. elegans, although some important differences exist. In the S. pombe system, an RdRP complex is proposed to convert RNA pol II-derived single strand RNA, generated at centromeric repeats, to dsRNA; components of the RdRP complex include RdP1 (RdRP), Hrr1 (helicase), and Cid12 (poly(A) polymerase). DsRNA is processed by Dicer to form siRNAs, which seed the assembly of an RNA-induced transcriptional silencing complex (RITS) whose components include Ago (Argonaute protein), Chp1 (chromatin-binding protein), and Tas3 (a protein responsible for ‘‘spreading’’ H3K9me2 marks along the chromosome). RITS in turn is hypothesized to recruit chromatin-modifying enzymes, for example, Clr4 HMTase (Buhler and Moazed, 2007; Buhler et al., 2007; Cam et al., 2005; Iida et al., 2008; Zhang et al., 2008). In this system, Chp1 must physically bind existing H3K9me2 marks in order for the machinery to deposit additional H3K9me2 marks on adjacent nucleosomes (Schalch et al., 2009). Although the details are unclear, it is hypothesized that activity of the siRNA pathway establishes a ‘‘self-amplifying’’ loop at centromeric DNA, leading to further recruitment of HMTase and further deposition of H3K9me2 marks (Buhler et al., 2007; Iida et al., 2008; Schalch et al., 2009; Zhang et al., 2008; see also Buhler and Moazed, 2007). In contrast to this mechanism, Dicer is not required for meiotic silencing in C. elegans (Maine et al., 2005), presumably because EGO-1-dependent 22G RNAs are generated in a Dicer-independent manner (Gu et al., 2009). Recent studies have implicated ncRNAs—both large and small—in heterochromatin assembly in mammals. A mammalian RdRP has recently been identified (Maida et al., 2009), and antisense transcripts are implicated in epigenetic regulation in mouse (Yu et al., 2008) and humans (Han et al., 2007; Morris et al., 2008). For example, Yu et al. (2008) report that inappropriate production of antisense transcripts due to mutation can result in silencing of genes that should be active, for example, tumor suppressor genes. Hence, inappropriate production of antisense transcripts due to mutation may repress gene expression at the chromatin level, leading to tumor growth and perhaps other disease conditions. It is estimated that antisense RNAs may be present for the majority of mouse transcripts, hence antisense transcripts may normally promote chromatin-based silencing of some genes. The mechanism of heterochromatin formation in this case is not understood, except that Dicer is not required, and therefore microRNA and other small RNAs whose biogenesis requires Dicer activity are not involved. Recent work in mammals has also demonstrated the association of chromatin-modifying complexes, such as PRC2 and an H3K9me2 methyltransferase complex called G9, with large intergenic noncoding (linc) RNAs (Khalil et al., 2009; Nagano et al., 2008; Ponting et al., 2009; Zhao et al., 2008). At present,

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the relative importance of different classes of ncRNAs in recruiting chromatin modifiers to specific genomic sites is unclear. However, it seems likely that small RNA-mediated mechanisms of chromatin regulation in mammals will share some features with regulatory mechanisms being uncovered in C. elegans and S. pombe.

7. Conclusions and Future Prospects Many unresolved questions remain regarding the mechanism and function of meiotic silencing in C. elegans and other species. Outstanding issues include the mechanism for sensing chromosome targets (i.e., unpaired/unsynapsed chromosomes), mechanisms for targeting H3K9me2 to them, and the function of H3K9me2 enrichment on those chromosomes. In addition, it is important to understand how H3K9me2 enrichment on unpaired chromosomes is related to the basal H3K9me2 accumulation detected across the genome and the physiological H3K9me2 enrichment occurring in the XX germ line during late meiotic prophase.

7.1. Mechanism What characteristic of a chromosome makes it a target for H3K9me2 enrichment? Based on studies in C. elegans and other organisms, two likely possibilities are that the cell assesses homolog alignment and/or synapsis. For example, the histone modifying machinery might be recruited via activity of a factor that is associated with unpaired or unsynapsed chromosomes but lost upon pairing or synapsis. Alternatively, it might be excluded via the activity of a factor that associates with chromosomes during pairing or synapsis. Recent evidence suggests that the XX and XO germ lines may use (at least partially) different mechanisms to identify chromatin for H3K9me2 accumulation (A. Fedotov and W.G. Kelly, personal communication). It is important to resolve the mechanistic details of how the EGO-1/ CSR-1 siRNA pathway, RHA-1, and other factors implicated in H3K9me2 regulation in fact act to target MET-2 activity to unpaired chromosomes. For example, does the siRNA machinery act directly on chromatin as is the case for centromeric regulation in S. pombe? Which component(s) confer specificity with respect to the target sites? How are the activities of these different factors integrated? A related question is how the mechanisms of basal and physiological H3K9me2 deposition are related to meiotic silencing. Based on its mutant phenotype (general loss of H3K9me2), RHA-1 may promote MET-2 activity, perhaps functioning as a component of the MET-2 complex. Are the ectopic H3K9me2 foci

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observed in csr-1, ekl-1, and drh-3 mutants the result of upregulated modification of what are normally sites of basal or physiological H3K9me2?

7.2. Function A central remaining question is how H3K9me2 modifications function at target sites. One strategy to address this question is to identify those targets and determine how loss of H3K9me2 (e.g., in an ego-1 mutant) might alter their expression—if indeed those sites are within coding regions. According to recent reports, EGO-1, DRH-3, and EKL-1 are responsible for the biogenesis of different classes of 22G-RNAs, one of which functions in posttranscriptional gene silencing (of transposons, pseudogenes, etc.) and another of which functions in mitotic chromosome segregation (Claycomb et al., 2009; Gu et al., 2009). The former class of siRNA acts as guide molecules for RISC complexes containing a set of partially redundant WAGO Argonautes, whereas the latter class of siRNA guides RISC complexes containing CSR-1/Argonaute (Claycomb et al., 2009; Gu et al., 2009). One hypothesis is that CSR-1 activity, targeted by EGO-1-generated siRNA, guides MET2 to unpaired chromosomes and/or prevents MET-2 from acting on paired chromosomes. Ectopic H3K9me2 observed in csr-1, drh-3, and ekl-1 mutants may reflect the inappropriate interaction of EGO-1-generated siRNAs with another RISC/Argonaute complex that normally is responsible for basal and/ or physiological H3K9me2 accumulation on autosomes. Critical to understanding the role of the chromatin-based meiotic silencing observed in animals is to determine the lower size limit for regions targeted by meiotic silencing in each species. For example, if regulation of individual genes is important, then meiotic silencing may act at the level of individual genes. If establishing chromatin structure across the chromosome is important, then meiotic silencing may act on a larger scale, targeting transcribed and nontranscribed regions. C. elegans has a robust posttranscriptional mechanism for repressing transposon activity, hence there may not be much selection pressure to repress transposons at the transcriptional level. Does meiotic silencing function in speciation? We know that heterologous pairing in C. elegans leads to incomplete synapsis, and that H3K9me2 is elevated on regions lacking a synaptonemal complex. It could be informative to determine whether meiotic chromosomes are enriched for H3K9me2 in interspecies hybrids, in particular males. Does elevated autosomal H3K9me2 correlate with male sterility in these cases? The answer may be yes: evidence from studies in Neurospora indicates that hybrid sterility can be partially alleviated when meiotic silencing is impaired (Shiu et al., 2001). On the flip side, evidence from mammals suggests that (at least some) components of the meiotic silencing machinery are limiting such that meiotic silencing of the X chromosome is less efficient when multiple unsynapsed regions are present (Homolka et al., 2007; Kouznetsova et al., 2009).

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ACKNOWLEDGMENTS The author is grateful to the following colleagues for discussions, sharing unpublished data, and comments on the manuscript: Jackie Arico, Michael Cosgrove, Alex Fedotov, David Greenstein, Jonathan Hodgkin, Bill Kelly, and Tim Schedl. The Maine laboratory receives support from the National Science Foundation, the National Institutes of Health, and Syracuse University.

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Lipid Rafts, Caveolae, and Their Endocytosis Patrick Lajoie* and Ivan R. Nabi† Contents 1. Introduction 2. The Lipid Raft 2.1. Origin of the concept 2.2. Defining raft domains 3. Caveolae 3.1. Caveolae and caveolin proteins 3.2. Cav1 and the regulation of caveolae formation 3.3. Cav1 domains outside of caveolae: Cav1 scaffolds 4. Raft-Dependent Endocytosis 4.1. Multiple rafts for multiple pathways 4.2. Regulation of raft-dependent endocytosis 4.3. Raft endocytosis and modulation of signaling cascades 5. Conclusion References

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Abstract Lipid rafts are plasma membrane microdomains enriched in cholesterol and sphingolipids that are involved in the lateral compartmentalization of molecules at the cell surface. Internalization of ligands and receptors by these domains occurs via a process defined as raft-dependent endocytosis. Caveolae are caveolin-1-enriched smooth invaginations of the plasma membrane that form a subdomain of lipid rafts. Endocytosis of rafts, including caveolar but also noncaveolar dynamin-dependent and dynamin-independent pathways, is characterized by its cholesterol sensitivity and clathrin-independence. In this review

* Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, USA { Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada International Review of Cell and Molecular Biology, Volume 282 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)82003-9

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2010 Elsevier Inc. All rights reserved.

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we will characterize lipid rafts and caveolae, their endocytosis and its regulation by the actin cytoskeleton, caveolin-1, dynamin, and cholesterol. Key Words: Caveolae, Lipid rafts, Endocytosis, Caveolin, Actin, Signaling. ß 2010 Elsevier Inc.

1. Introduction Caveolae were first identified in the 1950s and defined as smooth invaginations of the plasma membrane devoid of clathrin coat (Palade, 1953). These vesicular carriers represent a subdomain of plasma membrane lipid raft microdomains enriched in cholesterol and sphingolipids. Caveolae are abundant structures associated with expression of the caveolins, proteins that form the caveolar coat, and have been implicated in various biological processes such as endocytosis, transcytosis, and signal transduction. However, raft-dependent endocytosis is not limited to caveolae. Indeed, over the years, many other pathways have been characterized as caveolae-independent/raft-dependent endocytic pathways (Lajoie and Nabi, 2007). Defining caveolae and raft domains and the regulation of their formation and endocytosis by caveolin-1 (Cav1) and other factors is the subject of this review.

2. The Lipid Raft 2.1. Origin of the concept The observation that glycosphingolipids cluster in the Golgi apparatus before being sorted to the apical surface of polarized cells triggered the emergence of the lipid raft concept (Simons and van Meer, 1988). Further investigation later found that glycosphingolipid clusters are mostly insoluble in detergent at 4  C, therefore creating detergent-resistant membranes (DRMs). DRMs possess specific properties such as a light buoyant density on sucrose gradients and enrichment in both cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) (Brown, 1994). Indeed, until recently, most of the evidence for rafts as functional domains within the cell came from studies of the DRM association of proteins and the effect of cholesterol-modulating agents on these interactions. However, the use of detergents to identify raft-associated proteins is controversial (Munro, 2003). It has been shown that different detergents are unlikely to reflect the same aspects of membrane organization such that the effect of a given detergent may vary according to cell type (Schuck et al., 2003). Also this approach cannot provide information regarding the preexisting

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organization in the multicomponent cell surface, nor a quantitation of its physical characteristics (Rao and Mayor, 2005). While DRMs are still thought by some to be able to give valuable information about raft heterogeneity (Pike, 2004), others do not share that opinion (Lichtenberg et al., 2005). Efforts have been made to develop new technologies to characterize raft behavior without having to use detergent extraction; however, these approaches are also rife with potential problems (Lommerse et al., 2004; Shaw, 2006). Glycosphingolipid- and cholesterol-rich lipid domain formation may be driven solely by lipid–lipid interactions. In giant unilamellar vesicles (GUVs) containing unsaturated phospholipid, sphingomyelin, and cholesterol, components spontaneously form two distinct phases (Silvius and Nabi, 2006). The liquid disordered state (Ld) is characterized by both highly flexible acyl chains and highly mobile lipid molecules. The liquid ordered state (Lo) contains tightly packed sphingomyelin and cholesterol molecules and presents more restricted motion (Brown and London, 1998). Other evidence for microdomain formation came from studies of GUVs where it was shown that membrane microdomains can form in lipid mixtures resembling those of the plasma membrane (Dietrich et al., 2001; Silvius, 2003). While segregation of raft domains in model membranes supports the raft hypothesis, difficulties linked to the characterization of membrane microdomains in biological membranes have led to various suggested models. It was proposed that proteins targeted to lipid rafts have a light buoyant density because they are surrounded by cholesterol and sphingolipids (referred to as a lipid shell). Lipid shells are thermodynamically stable structures that have an affinity for preexisting rafts. Hence, they target the protein they encase specifically to these membrane domains (Anderson and Jacobson, 2002). Although the shell is proposed to be stably associated with proteins, its lipid components may interact and even interchange with nonshell lipids (Jacobson et al., 2007). While the existence of a lipid shell can explain the low buoyancy of DRMs, the extent to which stable protein–lipid interactions in biological membranes exist and determine raft association remains to be determined.

2.2. Defining raft domains Estimates of the size of raft domains are highly variable and have included: 15 protein molecules by protein cross-linking (Friedrichson and Kurzchalia, 1998); 22  4 nm diameter by immunoelectron microscopy of Ras-anchored GFP in isolated plasma membrane sheets (Prior et al., 2003); 26  13 nm diameter by laser trap measurements of local diffusion (Pralle et al., 2000); <70 nM diameter and containing fewer than 50 molecules by FRET (Varma and Mayor, 1998); and larger transient confinement domains of hundreds of microns by single molecule tracking

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approaches (Dietrich et al., 2002; Schutz et al., 2000). FRET has been observed between lipid-modified raft-associated monomeric GFPs (Zacharias et al., 2002). However, FRET studies of the well-characterized raft component, GPI-APs, generated conflicting data as to whether they cluster within raft domains (Glebov and Nichols, 2004; Kenworthy and Edidin, 1998; Kenworthy et al., 2000; Varma and Mayor, 1998). The conflicting data could be reconciled if a small proportion of the GPI-APs were localized to small clusters (Glebov and Nichols, 2004; Kenworthy et al., 2000). Indeed, fluorescence anisotropy measurements show that the majority of cell surface GPI-APs are present as monomers and that 20–40% are present in small (<5 nm) clusters (Sharma et al., 2004b). Similarly, single particle tracking analysis of GPI-linked CD59 described rafts as small, unstable structures of only a few molecules with a halftime of less than 1 ms (Subczynski and Kusumi, 2003). Also, further evaluation of the spatial distribution of Ras-anchored GFP by electron microscopy supported the notion of a minimal raft cluster of three to seven protein molecules (Parton and Hancock, 2004). Recent studies using single particle tracking and stimulated emission depletion (STED) have described the cholesterol-sensitive transient anchoring of raft-associated sphingolipids and GPI-APs in domains (Chen et al., 2006; Eggeling et al., 2009). The STED studies showed that transient anchoring occurred for 10–20 ms in domains smaller than 20 nm (Eggeling et al., 2009). Lipid rafts are now universally defined as small (10–200 nm) heterogeneous membrane domains enriched in sphingolipid and cholesterol that are involved in various cellular processes (Pike, 2006). Using high spatial and temporal FRET microscopy, it was shown that GPI-AP clusters are organized on at least two length scales at steady state: the nanoscale (<10 nm) and the optically resolvable scale (>450 nm) (Goswami et al., 2008). The larger complex could correspond to the larger raft domains proposed earlier (Simons and van Meer, 1988). The formation of these cholesterol-sensitive clusters is regulated by cortical actin indicating an active role for the actin cytoskeleton in the dynamics of plasma membrane domain formation (Goswami et al., 2008). Recently, fluorescence photoactivation localization microscopy (FPALM) has localized raft-associated hemagglutinin (HA) to irregular clusters ranging from 40 nm to many mms (Hess et al., 2007). Laminin treatment has been shown to induce the dystroglycandependent clustering of the raft-associated GM1 ganglioside into large raft macrodomains. Restricted GM1 diffusion in these domains requiring the actin-associated dystroglycan complex supports a role for organization of raft domains by the extracellular matrix and the underlying actin cytoskeleton (Noel et al., 2009). T and B cell receptor signaling results in the formation of large signaling complexes whose interaction is cholesterol-sensitive and that are associated with DRMs (Cheng et al., 2001; Holowka et al., 2005; Montixi et al., 1998). Raft components such as GM1 are observed by

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fluorescence microscopy to cluster together upon receptor ligation in T cells (Gaus et al., 2005; Janes et al., 1999; Viola and Lanzavecchia, 1999) and mast cells (Thomas et al., 1994), a process that involves the actin cytoskeleton (Chen et al., 2007). Extracellular matrix, receptor–ligand interactions and intracellular actin-dependent remodeling can therefore result in the reorganization of raft domains into large optically resolvable structures. The presence of lipid rafts is not limited to the plasma membrane of the cell. Endoplasmic reticulum (ER) proteins erlin1 and the sigma-1 receptor are found in raft fractions localizing lipid raft microdomains to the ER (Browman et al., 2006; Hayashi and Su, 2003). Due to the increase in sphingolipids and cholesterol along the secretory pathway, lipid rafts are believed to become more abundant in the Golgi complex (Eberle et al., 2002). Cholesterol-sensitive lipid raft microdomains have also been identified on the membrane of endosomes and phagosomes (Dermine et al., 2001; Gagescu et al., 2000; Sobo et al., 2007). Cav1 is also targeted to lipid droplets where it regulates cellular cholesterol homeostasis (Fujimoto et al., 2001; Ostermeyer et al., 2001; Pol et al., 2001). Understanding raft domain heterogeneity and organization in the plasma membrane, not to mention their relationship to intracellular raft domains therefore clearly remains a complex and evolving field of study. We can expect the definition of lipid rafts to undergo continuous change as the technology available for their characterization evolves.

3. Caveolae 3.1. Caveolae and caveolin proteins Caveolae, plasma membrane invaginations of 60–80 nm in diameter, were first identified in the 1950s by electron microscopy (Palade, 1953). Caveolae are expressed in various tissues and cell types such as smooth muscle, fibroblasts, endothelial cells, and adipocytes. The functions of caveolae are diverse and include endocytosis, transcytosis, potocytosis, calcium signaling, and regulation of various signaling events (Parton and Simons, 2007). The major constituent of caveolae is the protein Cav1 (Rothberg et al., 1992). Cav1 expression in cells devoid of caveolae, such as lymphocytes and CaCo-2 cells, is sufficient to induce caveolae (Fra et al., 1995; Lipardi et al., 1998; Vogel et al., 1998). Mammalian cells express two other isoforms of caveolin (Cav2 and Cav3) with Cav3 being muscle specific. Cav3 is more closely related to Cav1, 65% identical and 85% similar, while Cav2 is 38% identical and 58% similar to Cav1 (Scherer et al., 1996; Tang et al., 1996). While Cav1 and Cav3 are predominantly expressed on the plasma membrane, Cav2 is localized in the Golgi apparatus and is targeted to the

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cell surface and caveolae only upon formation of heterooligomers with Cav1 (Breuza et al., 2002; Li et al., 1998; Mora et al., 1999; Parolini et al., 1999). All three caveolin proteins share a common topology with N and C termini in the cytoplasm and a long hairpin transmembrane domain (Fig. 3.1). The structure of the three caveolin proteins is almost the same: the amino terminal domain comprises the first 101 amino acid residues in Cav1a and 70–86 residues in Cav1b, Cav2, and Cav3. The transmembrane domain comprises 33 amino acids and the C-terminal part of the protein contains 43–44 amino acids (Fig. 3.2) (Scherer et al., 1995; Williams and Lisanti, 2004). These structural differences impact on protein behavior. Indeed, only Cav1a contain the tyrosine 14 (Y14) residue required for its Src-dependent phosphorylation (Glenney, 1989; Li et al., 1996). Cav1 can also be phosphorylated on serine residue 80 (Ser80) that allows binding of the protein to the ER membrane thereby regulating its entry into the secretory pathway (Schlegel et al., 2001). Cav1 palmitoylation on cysteine residues 133, 143, and 156 allows the protein to oligomerize (Dietzen et al., 1995; Monier et al., 1996). Cav2 is phosphorylated on tyrosine 19 and 27 (Y19 and Y27) (Wang et al., 2004). Cav2 phosphorylation on Y19 upon treatment with insulin increased interaction between pY19Cav2 and pERK while phosphorylation of Cav2 on Y27 prolonged activation of the insulin receptor and promoted nuclear translocation of STAT3 (Kwon and Pak, 2009; Kwon et al., 2009a,b). Cav2 is also phosphorylated

Extracellular space

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Figure 3.1 Cav1 topology. Cav1 is a 178 amino acid protein inserted into the inner leaflet of the plasma membrane by a hairpin domain resulting in both N- and C-terminal domains facing the cytoplasm. The scaffolding domain (CSD) contains the amino acids 82–101. Cav1 can be phosphorylated on Tyr14 and Ser80.

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Figure 3.2 Caveolin isoforms. Cav1 is present in two different isoforms: Cav1a and Cav1b. Cav1a contains the full 178 caveolin amino acids while Cav1b is limited to amino acids 32–178. Both isoforms are phosphorylated on S80 and palmitoylated on cysteines C133, C143, and C146. Only Cav1a is phosphorylated on Y14. Cav2 contains amino acids 1–162 ( 38% identical) of Cav1 and is phosphorylated on Y19, Y27, S23, and S36. Cav3 contains amino acids 1–151 (65% identical) of Cav1 and is palmitoylated on cysteines C133, C143, and C146.

on Ser23 and Ser36 in a process regulated by protein targeting to lipid raft domains (Sowa et al., 2008). Caveolin proteins exist as monomers in the Golgi apparatus (Pol et al., 2005) but once in the secretory pathway upon transport to the plasma membrane, they form high-molecular-weight oligomers (Ren et al., 2004). The first step of oligomerization occurs in the ER not long after synthesis of the protein (Monier et al., 1996). Newly synthesized Cav1 is then transported to the Golgi apparatus and at this step, Cav1 association with DRMs has not yet occurred (Pol et al., 2005). Along their subsequent transport through the secretory pathway, Cav1 oligomers form complexes with DRMs, a characteristic of plasma membrane caveolins (Scheiffele et al., 1998). Exit of Cav1 from the Golgi complex is accelerated upon addition of cholesterol (Pol et al., 2005) and inhibited upon glycosphingolipid depletion (Cheng et al., 2006), supporting a role for recruitment to raft domains in Cav1 transport from the Golgi to the cell surface.

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Using Cav1–GFP fusion protein, it was shown that Cav1 transits directly from the Golgi complex to the plasma membrane (Tagawa et al., 2005). Based on the size of the structure exiting the Golgi, it was proposed that Cav1 oligomerizes and associates with cholesterol and glycosphingolipids to form exocytic structures similar to mature plasma membrane caveolae. These structures were named ‘‘exocytic caveolar carriers’’ (Parton and Simons, 2007). However, the identification of these carriers does not necessarily identify the site of initial formation of caveolae. The slow net transit of caveolin out of the Golgi might be sufficient to allow Cav1 to reach a certain threshold and allow oligomerization and association with lipid rafts (Parton et al., 2006). Certain anti-Cav1 antibodies selectively recognize the Golgi pool of Cav1 but not the protein at the plasma membrane, indicative of a change in Cav1 conformation during its transport to the cell surface. Cholesterol depletion is able to restore this reactivity, suggesting that Cav1 association with rafts may be responsible for differential antibody recognition (Pol et al., 2005).

3.2. Cav1 and the regulation of caveolae formation The ability of Cav1 to oligomerize is a key determinant of its ability to form caveolae. Cav1 forms higher molecular weight oligomers consisting of 14–16 Cav1 molecules (Monier et al., 1996; Sargiacomo et al., 1995). The 41 residue oligomerization domain preceding the putative transmembrane domain mediates homooligomerization of Cav1 and is required for the formation of large oligomers (Sargiacomo et al., 1995; Schlegel et al., 2000). It is likely that Cav1 oligomerization facilitates the formation of Cav1-enriched microdomains at the cell surface. Tryptophan residues at the membrane interface could further increase cholesterol recruitment and insertion of the scaffolding domain into the membrane, achieving greater expansion of the cytoplasmic leaflet of the caveolar bulb (Parton et al., 2006). Cav2 has also been implicated in caveolae formation, and its serine phosphorylation found to modulate the formation of deep surface attached caveolae versus nonattached caveolar vesicles (Fujimoto et al., 2000; Scheiffele et al., 1998; Sowa et al., 2003). However, while the Cav1 null mouse shows loss of caveolae, caveolae are still present in the Cav2 mouse defining an essential role for Cav1 in caveolae formation in nonmuscle tissues (Drab et al., 2001; Razani et al., 2001, 2002; Zhao et al., 2002). In muscle, Cav3 is essential for caveolae formation and Cav3 mutations and loss of Cav3 and caveolae results in a dystrophic phenotype (Galbiati et al., 2000; McNally et al., 1998; Minetti et al., 1998). Cav1 expression at the plasma membrane is not sufficient to induce caveolae formation. Cholesterol extraction has been extensively shown to disrupt caveolae at the plasma membrane (Rothberg et al., 1992). Threshold levels of cholesterol are required for caveolae formation and Cav1

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incorporation into raft domains at the plasma membrane is a critical determinant of caveolae formation (Breuza et al., 2002; Hailstones et al., 1998). Cav1 binds cholesterol via a putative cholesterol-binding domain and Cav1 has been implicated in cholesterol trafficking and efflux (Fielding and Fielding, 1995; Murata et al., 1995). Interaction of Cav1 with cholesterol may be required for the reorganization of Cav1 oligomers to form invaginated vesicles (Rothberg et al., 1992). Cav1 tyrosine phosphorylation (Y14) has also been recently implicated in caveolae formation in epithelial cells (Orlichenko et al., 2006). Importantly, another protein, PTRF/cavin-1, is also needed for the formation of caveolae (Fig. 3.3). Cells lacking this protein, but expressing Cav1, present diffuse cell surface Cav1 labeling and fail to express caveolae (Hill et al., 2008). Another member of this protein family, SDPR/cavin-2, is also required for caveolae formation and its overexpression results in the formation of enlarged caveolae and elongated caveolae-derived tubules (Hansen et al., 2009). SRBC/cavin-3 is not required for caveolae formation but does impact on the budding and trafficking of caveolar vesicles (McMahon et al., 2009). A fourth member of the cavin family, MURC/ cavin-4, is muscle specific and regulates caveolae formation in muscle (Bastiani et al., 2009). The cavins form a caveolin-associated protein complex (Bastiani et al., 2009; Hansen et al., 2009) and how this protein complex functions to regulate caveolae expression and caveolin function is a field of active investigation that should provide important new insight into caveolae function (Nabi, 2009).

3.3. Cav1 domains outside of caveolae: Cav1 scaffolds The ability of various modulators and interactors to impact on Cav1 ability to form caveolae indicates that formation of caveolae is a highly complex and regulated cellular process. At the same time, the fact that expression of Cav1 is not sufficient for caveolae formation indicates that Cav1 is present in cells lacking caveolae and should necessarily have functions outside of caveolae. Indeed, there is growing evidence in the literature that Cav1 regulates cellular processes outside of caveolae (Head and Insel, 2007; Lajoie et al., 2009a; Parton and Simons, 2007) Cav1 contains a scaffolding domain that has the ability to bind various classes of signaling proteins such as G-protein subunits, receptor and nonreceptor tyrosine kinases, endothelial nitric oxide synthase (eNOS), and small GTPases (Okamoto et al., 1998; Parton and Simons, 2007). Indeed, interaction of Cav1 with a variety of signaling receptors results in inhibition of their signaling potential (Goetz et al., 2008b). The extent to which this regulatory activity of Cav1 requires expression of caveolae is unclear. Cav1 can regulate epidermal growth factor receptors (EGFR) diffusion and signaling and CT-b diffusion and endocytosis at levels below the threshold

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Albumin SV40

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Caveolin-1 positive Dynamindependent Cholesteroldependent Cholesterol

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Figure 3.3 Raft-dependent endocytosis encompasses various pathways. Cholesterolsensitive internalization of lipid rafts can be classified into three major pathways. These include dynamin-dependent endocytosis of caveolae or noncaveolar vesicular carriers as well as dynamin-independent endocytosis via noncaveolar tubular intermediates. Raft-dependent internalization of specific receptors or molecules may occur via multiple pathways, each involving different vesicular intermediates and regulated by specific endocytic machineries.

required for caveolae formation (Lajoie et al., 2007, 2009a). Cav1 can also restrict CT-b diffusion on the endosomal membrane (Pelkmans et al., 2004). Interestingly, endothelial-specific overexpression of Cav1 impaired VEGF signaling without impacting on caveolae number (Bauer et al., 2005). Tyrosine phosphorylated Cav1 has been implicated in focal adhesion organization and dynamics (Gaus et al., 2005; Goetz et al., 2008a; GrandeGarcia et al., 2007; Joshi et al., 2008). These studies describing caveolaeindependent functions for Cav1 led to the definition of Cav1 scaffolds, a

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term to describe oligomeric Cav1 domains present at the cell surface that do not correspond to invaginated caveolae (Lajoie et al., 2009a). Cav1 and phosphorylated Cav1 may define functionally distinct scaffold domains (Lajoie et al., 2009a). Recently, it was shown that Cav1 and Cav2 form a plasma membrane scaffold containing 15–25 caveolins molecules that can associate with cavins to form caveolae (Hayer et al., 2009). The identification of the cavins may facilitate further study of the selective functions of caveolae and Cav1 scaffolds. Caveolae therefore represent a subdomain of lipid raft microdomains that are stabilized by caveolin proteins. Lipid rafts are defined as cholesteroland sphingolipid-enriched membrane domains and numerous studies have attempted the lipid characterization of caveolae and raft domains. Early immunoelectron microscopy studies identified GM1 enrichment in caveolae (Schnitzer et al., 1995). However, GM1 has also been localized to raft domains that associate with nascent clathrin-coated pits (Puri et al., 2005). Analysis of purified caveolae fractions found that the glycosphingolipid GD3 was highly enriched in caveolae, GM3, GM1, and GD1a present inside and outside caveolae membranes and various other glycosphingolipids localized exclusively outside the caveolae fraction (Ortegren et al., 2004). By confocal microscopy, distribution of GM1 and another wellcharacterized raft marker, GPI-APs, do not overlap with Cav1 (Lajoie et al., 2009b; Noel et al., 2009). Another raft marker, flotillin, defines noncaveolar rafts that can contain GPI-APs and GM1 (Fernow et al., 2007; Glebov et al., 2006; Noel et al., 2009; Stuermer et al., 2001). Raft-associated lipids and proteins therefore exhibit a heterogeneous distribution between caveolar and noncaveolar raft domains.

4. Raft-Dependent Endocytosis 4.1. Multiple rafts for multiple pathways Internalization of molecules and ligand via clathrin-coated vesicles has been extensively studied and well characterized (Benmerah and Lamaze, 2007; Roth, 2006). However, over the years, many other endocytic routes have been described and do not rely on the formation of clathrin-coated pits. Characterization of these alternate pathways has allowed a better understanding of the complexity of the endocytic process in mammalian cells (Fig. 3.3). Many of these clathrin-independent pathways share an important characteristic: their sensitivity to cholesterol depletion. These pathways rely on internalization of dynamic lipid-rich carriers and are therefore regrouped under the appellation raft-dependent endocytosis.

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4.1.1. Caveolae Although many raft-dependent pathways have been shown to not rely on the presence of caveolin proteins, various molecules or ligand are internalized via caveolae. In endothelial cells, caveolae represent the major route of transcytosis-transporting albumin (Tiruppathi et al., 1997). The albumin docking protein gp60 localizes to caveolae in endothelial cells and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles (Minshall et al., 2000). Modification of plasma albumin on tyrosine residues generates nitrated albumin (NOA) that can be transported across the vascular endothelium via caveolae-dependent transcytosis (Predescu et al., 2002). Gbg activation of Src kinase signaling induced caveolaemediated endocytosis and transendothelial albumin transport via transcytosis (Shajahan et al., 2004b) in a process that involves dynamin-2 phosphorylation (Shajahan et al., 2004a). Caveolae-dependent transcytosis of albumin is dependent on the F-actin cross-linking protein filamin A (Sverdlov et al., 2009). Cav1 phosphorylation also regulates H2O2-induced pulmonary vascular albumin hyperpermeability via transcytosis (Sun et al., 2009). Increased endothelial caveolae leading to transcytosis of plasma proteins is associated with blood–brain barrier (BBB) breakdown and it is suggested that Cav1 phosphorylation may be one of the key factors involved in this kind of brain injury (Nag et al., 2009). Using intravital microscopy, it was shown that caveolae successfully transport antibody against aminopeptidase P (APP) from the blood across the endothelium into lung tissue (Oh et al., 2007). Dynamin is a protein involved in the fission of some endocytic vesicles from the plasma membrane (Henley et al., 1999). Caveolae budding from the plasma membrane and subsequent internalization requires dynamin II which is localized at the neck of the vesicle (Oh et al., 1998). Intersectin is another protein that has been shown to be involved in the fission of nascent caveolae from the plasma membrane (Predescu et al., 2007). It was shown that the intersectin is part of a protein complex containing dynamin and SNAP-23 and is localized at the neck of the budding vesicle (Predescu et al., 2003). It was recently shown that intersectin-2L, a guanine nucleotide exchange factor for Cdc42, regulates caveolae endocytosis by regulating actin polymerization and remodeling at the plasma membrane (Klein et al., 2009). In nonendothelial cells, fluorescent glycosphingolipid analog, BODIPYlactosylceramide (LacCer) has been shown to be specifically internalized via a caveolae-dependent pathway (Sharma et al., 2003). Caveolae-dependent endocytosis has been shown to be regulated by both gangliosides and b1-integrin expression (Singh et al., 2010). In primary adipocytes, the insulin receptor undergoes rapid internalization via caveolae (Fagerholm et al., 2009). SV40 is endocytosed by caveolae and subsequently targeted to a Cav1 positive endosomal compartment: the caveosome (Pelkmans et al., 2001). However, more recent studies suggest that SV40 is internalized

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predominantly via a noncaveolar, dynamin-independent route (Damm et al., 2005; Ewers et al., 2010). Similarly, early studies demonstrated that CT-b may be internalized in a process that involves caveolae (Henley et al., 1998; Oh et al., 1998; Parton et al., 1994). However, more recent studies suggest that the major raft-dependent endocytic route for CT-b is noncaveolar (Kirkham et al., 2005; Lajoie et al., 2009b). The recent demonstration that SV40 binding to GM1 can induce membrane invaginations suggests that interaction of SV40 and potentially cholera toxin with GM1 may induce membrane curvature that promotes endocytic uptake (Ewers et al., 2010). This process does not require Cav1; however, tubule formation by Shiga toxin, whose receptor is the ganglioside GM3, has been shown to derive from caveolae and be modulated by SDPR/cavin-2 defining a potential role for Cav1 and cavins in raft-dependent endocytic processes (Hansen et al., 2009; Romer et al., 2007). Therefore, at least in nonendothelial cells, the role of caveolae as a major endocytic carrier is questionable and other raftdependent endocytic pathways, independent of caveolae, may play a more predominant role in endocytosis. Most studies of caveolae endocytosis are based on live cell imaging techniques using Cav1 fluorescent protein chimeras. While useful, fluorescent proteins must be used with care and data analyzed with caution (Snapp, 2009). Cav1–GFP has been shown to induce caveolae and may favor localization of the protein of interest into caveolae. Furthermore, in transiently transfected overexpressing cells, it is unclear how much of the expressed Cav1–GFP protein is actually in caveolae and studies of Cav1 dynamics, and presumably caveolae, may actually be visualizing noncaveolar Cav1–GFP. Photobleaching experiments have revealed that Cav1 is highly stable at the plasma membrane (Thomsen et al., 2002), questioning their role in constitutive endocytosis (Hommelgaard et al., 2005). In contrast, clathrin-coated pits, for instance, are highly mobile and the entire pool of cell surface clathrin-coated vesicles is turned over within 6 min (De Bruyn and Cho, 1987). Indeed, it was proposed that Cav1 may stabilize raft domains at the plasma membrane, thus acting as a negative regulator of raft-dependent endocytosis (Lajoie and Nabi, 2007; Nabi and Le, 2003). Caveolar endocytosis may represent a minor, regulated pathway restricted to specific ligands. Internalization of ligands via caveolae may depend on various factors, such as caveolin, cavins, and ganglioside and cholesterol levels. 4.1.2. Noncaveolar dynamin-dependent endocytosis Autocrine motility factor (AMF) is another ligand that has been shown to be localized to caveolae and internalized via a dynamin-dependent pathway (Benlimame et al., 1998; Le et al., 2002). Internalization of this protein is enhanced in cells expressing reduced Cav1 levels, indicating that it is internalized via a noncaveolar dynamin-dependent pathway (Kojic et al.,

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2007, 2008; Le et al., 2002). Formation of dynamin-dependent smooth plasma membrane vesicles occurs both in the presence or absence of caveolins, indicating that dynamin regulates the formation of raft-derived endocytic carriers independently of Cav1 (Le et al., 2002). Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes and is coupled to their association with DRMs. This raft-dependent pathway requires dynamin and is specifically regulated by Rho family GTPases (Lamaze et al., 2001). Raft-dependent internalization of CT-b from the plasma membrane has also been shown to occur via a noncaveolar dynamin-dependent process (Lajoie et al., 2009b). Flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both GPI-linked proteins and CT-b. siRNA against flotillin-1 switched the internalization of CT-b from a dynamin-dependent pathway to a dynamin-independent endocytic route (Glebov et al., 2006). 4.1.3. Noncaveolar dynamin-independent endocytosis Evidence for a clathrin-independent noncaveolar pathway (or clathrinindependent carrier (CLIC)/GPI-AP-enriched early endosomal compartment (GEEC) pathway) came from analysis of cells expressing a temperature-sensitive mutant form of dynamin. Interestingly, blocking clathrin (and presumably caveolae)-mediated endocytosis caused an initial block in fluid phase internalization followed by a gradual compensatory increase. After 30 min fluid phase uptake returned to the same level as before the temperature shift while receptor-mediated endocytosis remained blocked (Damke et al., 1994). Pinocytic fluid phase uptake of multiple GPI-APs was also shown to rely on a noncaveolar raft endocytic pathway independent of dynamin and RhoA (Sabharanjak et al., 2002). This pathway is regulated by ARF1 and CDC42 (Kumari and Mayor, 2008). More recently, it was shown that the dynamin-independent internalization of CT-b in mouse embryonic fibroblasts occurred predominantly via uncoated tubular or ring-shaped vesicular carriers containing GPI-APs but not Cav1 (Kirkham et al., 2005). Dynamin-independent raft pathways also include a noncaveolar endocytic route for the simian virus SV40 (Damm et al., 2005; Ewers et al., 2010). CT-b endocytosis via a dynamin-independent raft pathway was flotillin-1-dependent, suggesting that flotillin may define raft domains that internalize via this dynamin-independent route (Glebov et al., 2006). Cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling, and macropinocytosis potentially defining another raft-dependent dynamin-independent pathway (Grimmer et al., 2002; Schneider et al., 2007). Multiple raft-dependent endocytic pathways therefore exist reflecting the heterogeneity of lipid raft microdomains at the cell surface (Hancock,

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2006). Many pathways have been characterized and a number of raft endocytic ligands are internalized via different endocytic routes. This varies among cell types and is dependent on receptor expression, ligand concentration, and other factors. For example, transforming growth factor beta (TGFb) is internalized via both clathrin-coated pits and caveolae (Di Guglielmo et al., 2003). Stimulation with low doses of epidermal growth factor (EGF) leads to receptor internalization via clathrin-mediated endocytosis, while higher dose induces its internalization via a raft-dependent pathway (Sigismund et al., 2005). AMF can be internalized via clathrincoated pits as well as rafts in NIH-3T3 cells (Benlimame et al., 1998; Le et al., 2000). Internalization via different endocytic pathways is also a characteristic of CT-b. In different cell types, it can be internalized via both clathrin and raft-dependent endocytosis (Pang et al., 2004; Torgersen et al., 2001). Intriguingly, selectivity of GPI-APs for dynamin-independent, noncaveolar raft uptake appears to be related not to the lipid anchor but rather to steric considerations of the anchored protein (Bhagatji et al., 2009). This suggests that multiple elements, including relatively nonspecific ones such as steric bulk, can contribute to selection of the route of endocytosis.

4.2. Regulation of raft-dependent endocytosis 4.2.1. Negative regulation by Cav1 Cav1 is known for its role in vesicle formation and subsequent internalization of cargo via caveolae-mediated endocytosis. However, there is growing evidence that Cav1 can also act as a negative regulator of raft-dependent endocytosis (Lajoie and Nabi, 2007; Nabi and Le, 2003). Indeed, it was shown that Cav1 can reduce or inhibit CT-b internalization via raft pathways (Kirkham et al., 2005; Lajoie et al., 2009b; Le and Nabi, 2003). It was also shown that Cav1 overexpression inhibits AMF uptake (Kojic et al., 2007; Le and Nabi, 2003; Le et al., 2002), dysferlin uptake (HernandezDeviez et al., 2008), and raft-dependent integrin endocytosis (Vassilieva et al., 2008). Cav1 overexpression is also associated with inhibition of endocytosis of albumin in HeLa cells (Sharma et al., 2004a). Interestingly, the ability of Cav1 to inhibit endocytosis is not necessarily caveolae-associated supporting a role for Cav1 scaffolds in this process. In mammary tumor cells, Cav1 levels below the threshold required for caveolae formation is sufficient to reduce CT-b internalization (Lajoie et al., 2009a). Also, overexpression of Cav1 is responsible for inhibition of internalization of dysferlin without direct interaction with the protein (Hernandez-Deviez et al., 2008). These data support the idea of an indirect mechanism for the negative regulatory activity on Cav1. Presence or absence of Cav1 may modulate the protein and lipid composition of cell surface rafts, therefore regulating their endocytic potential. Raft endocytic

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ability may be regulated by a dynamic equilibrium between cholesterol, lipids, and protein within these domains. 4.2.2. The role of cholesterol Cholesterol is a major constituent of lipid rafts and its concentration at the plasma membrane generally regulates raft-dependent phenomena such as signaling and endocytosis. Cholesterol is one of the key factors determining long-range protein mobility at the cell surface (Kenworthy et al., 2004). Cholesterol can regulate caveolae formation and decreased total cellular cholesterol levels were associated with similar decreases in Cav1 protein concentration in cholesterol-depleted cells (Breuza et al., 2002; Hailstones et al., 1998). Cholesterol-modulating agents, including methyl-b-cyclodextrin, nystatin, and filipin, have been variously shown to inhibit both caveolae expression and raft-dependent endocytosis (Damm et al., 2005; Hailstones et al., 1998; Le et al., 2002; Parpal et al., 2001; Schnitzer et al., 1994; Sharma et al., 2004a). Cav1 interacts directly with cholesterol (Fielding et al., 2002; Murata et al., 1995) and cholesterol levels in lipid raft fractions obtained from Cav1 expressing cells were three- to fourfold higher than in matched cells lacking Cav1 (Smart et al., 1996). When cholesterol exit from the ER is inhibited, Cav1 accumulates in the ER identifying a role for cholesterol in Cav1 trafficking to the plasma membrane (Smart et al., 1996). Increased total cholesterol by acute treatment with cyclodextrin/cholesterol or by alteration of growth conditions was shown to significantly stimulate caveolar endocytosis (Sharma et al., 2004a). In heterokaryons containing GFP- and RFP-tagged Cav1, individual caveolar domains undergo little exchange of Cav1. However, when the cells were subjected to transient cholesterol depletion, the caveolar domains were found to exchange Cav1 (Tagawa et al., 2005). Cholesterol therefore represents a major regulator of raft endocytosis and critically impacts on Cav1 expression, dynamics, and ability to form caveolae. 4.2.3. The actin cytoskeleton Another key regulator of raft-dependent endocytosis is the actin cytoskeleton. In fact, it was shown that complete depolymerization of the actin cytoskeleton with latrunculin A induces rapid and massive movements of caveolin-positive structures toward the centrosomal region of the cell (Mundy et al., 2002). This is in agreement with findings showing that caveolae are stable plasma membrane compartments stabilized by the actin skeleton. Indeed, using fluorescence recovery after photobleaching, it was shown that while intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were highly immobile (Thomsen et al., 2002). Cav1 can bind to actin-associated filamin via the N-terminal region of Cav1 and the C terminus of filamin close to the filamin-dimerization domain

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(Stahlhut and van Deurs, 2000). Interestingly, after binding to caveolae, SV40 induced transient breakdown of actin stress fibers. Actin was then recruited to SV40 positive caveolae as actin patches (Pelkmans et al., 2002). More recently, TIRF microscopy was used to show that reversible caveolae budding is limited to the subplasma membrane region by the underlying actin cytoskeleton (Tagawa et al., 2005). Cell detachment from substrate induces clearance of rafts from the cell surface in a Cav1-dependent endocytic process that requires F-actin (Balasubramanian et al., 2007; del Pozo et al., 2005). However, disruption of the actin cytoskeleton by cytochalasin D in A431 cells inhibited alkaline phosphatase uptake via caveolae (Parton et al., 1994). The actin cytoskeleton was also shown to be a critical regulator of the internalization of the antigen–BCR complex (Caballero et al., 2006). The bacteria Porphyromonas gingivalis is also thought to be internalized via a pathway requiring both rafts and an intact actin skeleton (Tsuda et al., 2008). Actin depolymerization also induces internalization of tight junction proteins via a caveolae-dependent pathway (Shen and Turner, 2005). Interestingly, when treated with PDGF, 23 proteins show increased raft localization including cytoskeleton proteins such as actin (MacLellan et al., 2005). Using TIRFM, mild cholesterol depletion was found to perturb actin polymerization by preventing CDC42 activation and thereby inhibiting endocytosis of GPI-APs through the GEEC pathway (Chadda et al., 2007). Raft domains therefore function together with the actin cytoskeleton to regulate raft endocytosis.

4.3. Raft endocytosis and modulation of signaling cascades Many proteins are localized to lipid rafts and these domains have been described as platforms for cellular signaling (Hancock, 2006; Simons and Gruenberg, 2000). Indeed, a quantitative proteomic approach has shown that many signaling molecules are enriched in rafts (Foster et al., 2003). Cav1 possesses a scaffolding domain implicated in the sequestration of receptors and signaling molecules (Liu et al., 2002; Okamoto et al., 1998). In quiescent fibroblasts, EGF and EGFR are initially concentrated in caveolae but rapidly move out of this membrane domain after stimulation with ligand, supporting the idea that caveolae represent a negative regulatory domain (Mineo et al., 1999). EGF signaling was inhibited in senescent cells expressing higher Cav1 levels (Park et al., 2000). In mammary tumor cells, competition exists between various cell surface domains for localization of EGFR and association with Cav1 scaffolds suppresses its signaling potential (Lajoie et al., 2007). A 60-amino acid-long sequence that is continuous with the transmembrane domain is sufficient to target both the transmembrane and cytoplasmic tails of EGFR to caveolae/rafts (Yamabhai and Anderson, 2002). Indeed, Cav1 can induce the

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sequestration of the receptor in a cholesterol-sensitive way (Matveev and Smart, 2002). Cav1 also inhibits EGFR-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3) (Zhang et al., 2000). However, EGFR was shown to be present in a caveolin-rich fraction from A431 cells, but failed to coimmunoprecipitate with caveolin, indicating that the Cav1 effect on EFGR signaling may be indirect (Waugh et al., 1999). Phosphorylated EGFR also did not coimmunoprecipitate with Cav1, arguing against a caveolar location for EGFR signaling (Waugh et al., 1999). Moreover, it is thought that cholesterol may regulate EGFR signaling independently of Cav1 by modulating the content of lipid rafts (Pike and Casey, 2002; Pike et al., 2005; Westover et al., 2003). Indeed, domain competition for recruitment of EGFR between lipid rafts and Cav1 scaffolds may be regulated by its activation state as well as glycosylation and recruitment to galectin lattice domains (Lajoie et al., 2007; Puri et al., 2005). Similarly, TGFbR internalization via clathrin-dependent endocytosis correlates with subsequent signaling events via Smad2 phosphorylation in EEA1-positive endosomes while its caveolae/raft-dependent internalization is associated with receptor degradation through binding to the smad7–smurf2 complex (Di Guglielmo et al., 2003). Signaling cascades also impact on raft-dependent endocytosis by regulating the internalization of various endocytic carriers. Inhibition of tyrosine phosphorylation using pharmaceutical agents blocks caveolae endocytosis while treatment of the cells with phosphatase inhibitor okadaic acid triggers endocytosis (Kirkham et al., 2005; Parton et al., 1994; Pelkmans et al., 2002). Treatment of the cells with albumin was shown to induce Cav1 phosphorylation and inhibition of this process was associated with reduced uptake of albumin via transcytosis (Tiruppathi et al., 1997). Inhibition of tyrosine phosphorylation using genistein inhibits uptake of AMF in various tumor cell lines (Kojic et al., 2007, 2008). The same results were described for internalization of lactosylceramide (Sharma et al., 2004a). While the initial recruitment of SV40 to caveolae was not dependent on tyrosine phosphorylation, tyrosine kinase inhibition prevented the dynamin-dependent release of caveolae from the membrane (Pelkmans et al., 2002). Albumin treatment induced Src activation and resulted in Src-dependent tyrosine phosphorylation of dynamin-2. The Y231F/Y597F dynamin2 mutant expression also resulted in impaired albumin and cholera toxin subunit B uptake and reduced transendothelial albumin transport (Shajahan et al., 2004a). How activation and regulation of endocytic and signaling events within raft domains regulate cellular processes is a subject of intense investigation that may contribute to a better understanding of the functional significance of raft domain localization and endocytosis at the plasma membrane.

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5. Conclusion Lipid rafts are cholesterol- and sphingolipid-rich membrane domains that vary from highly transient nanoscale domains to larger, more stable structures. Caveolin proteins are key regulators of lipid raft domain expression that induce the formation of invaginated caveolae but also noncaveolae scaffold domains that regulate raft function and endocytosis. Raft endocytosis is regulated not only by Cav1 but also by cholesterol and the actin cytoskeleton and may play critical roles in the regulation of cellular signaling.

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C H A P T E R

F O U R

New Insights into the Mechanism of Fibroblast to Myofibroblast Transformation and Associated Pathologies Mitchell A. Watsky,* Karl T. Weber,† Yao Sun,† and Arnold Postlethwaite‡,§ Contents 1. 2. 3. 4.

Introduction Fibroblasts and Intermediates Myofibroblasts Fibroblast to Myofibroblast Signaling 4.1. Transforming growth factor-beta (TGFb) 4.2. TGFb cooperative proteins and independent pathways 5. Mononuclear Cells in Peripheral Blood That Differentiate into Myofibroblasts 5.1. Historical perspective 5.2. Neofibrosis 5.3. Fibrocytes 5.4. Other monocyte-derived progenitors of fibroblast-like cells 6. Myofibroblast-Associated Pathologies 6.1. Myofibroblasts and cardiac repair following infarction 6.2. Systemic sclerosis 7. Summary References

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* Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, USA { Division of Cardiovascular Diseases, Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA { Division of Rheumatology, Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, USA } Veterans Affairs Medical Center, Memphis, Tennessee, USA International Review of Cell and Molecular Biology, Volume 282 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)82004-0

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2010 Elsevier Inc. All rights reserved.

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Abstract Myofibroblasts are a differentiated cell type essential for wound healing, participating in tissue remodeling following insult. Myofibroblasts are typically activated fibroblasts, although they can also be derived from other cell types, including epithelial cells, endothelial cells, and mononuclear cells. In most organ systems, cell signals initiated following tissue-specific insult or during the metastatic process lead to differentiation of fibroblasts or other precursor cells to the myofibroblast phenotype. In addition to their beneficial and necessary role in wound healing, myofibroblasts also contribute to a number of pathologies, primarily fibrotic processes and tumor invasiveness. This review explores both traditional and nontraditional concepts of myofibroblast differentiation in the cornea, skin, heart, and other tissues, as well as some of the pathologies associated with myofibroblast activities. Key Words: Cornea, Fibrocyte, Fibrosis, Heart, Lung, Systemic sclerosis, Scleroderma. ß 2010 Elsevier Inc.

1. Introduction Fibroblasts and myofibroblasts are found in all tissues of the body. Fibroblasts can sit as relatively silent cells, as in the adult cornea (corneal keratocytes), contributing minimally to the homeostasis of the surrounding tissue; or they can be active, as appears to be the case in cardiac tissue (Kohl et al., 2005). Myofibroblasts are typically activated fibroblasts, although they can also be derived from other cell types, including epithelial cells, endothelial cells, and mononuclear cells. They contribute to routine tissue remodeling and wound healing and also contribute to a number of pathologies, primarily fibrotic processes and tumor invasiveness. In most organ systems, cell signals initiated following tissue-specific insult or during the metastatic process lead to differentiation of fibroblasts or other precursor cells to the myofibroblast phenotype. We will explore both traditional and nontraditional concepts of myofibroblast differentiation in the cornea, skin, heart, and other tissues, as well as some of the pathologies associated with myofibroblast activities. While epithelial cells are an important contributor to the pool of myofibroblasts, particularly in cancer models (epithelial–mesenchymal transition or EMT), for reasons of brevity we have chosen not to discuss it in this review; instead we refer the reader to several recent reviews on EMT (Acloque et al., 2009; Kalluri and Weinberg, 2009; Thiery et al., 2009; Zeisberg and Neilson, 2009).

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2. Fibroblasts and Intermediates Fibroblasts are often named according to their cell shape, or ‘‘fibroblast morphology,’’ which is usually long and spindle-shaped. Fibroblasts typically lack a basement membrane, reside within the extracellular matrix, and may or may not have multiple cell processes, depending on the tissue in which they reside. In many tissues, such as the cornea, fibroblasts are functional during development, laying down a portion of the organ matrix, and become relatively quiescent in the steady-state adult tissue (Hay, 1979). In the cornea, the name ‘‘fibroblast’’ is actually given to an intermediate cell type whose differentiated state is between that of the quiescent keratocyte and the activated myofibroblast. It is actually the corneal keratocyte that is the functional fibroblast cell type in the cornea, although morphologically it is more dendrite-shaped than fibroblastic, whereas the intermediate ‘‘fibroblast’’ phenotype does indeed have the typical fibroblast morphology. In the cornea, as in other tissues, phenotype distinction between the different cell types is made not only by cell morphology but also by specific cell proteins that are either specifically expressed by the phenotype or expressed in greater or lesser quantities by the phenotype. In all tissues, the universal distinction between fibroblasts and myofibroblasts in vivo is the presence of alpha smooth muscle actin (aSMA) in myofibroblasts. In cultured cells, fibroblasts may also have small quantities of aSMA, although myofibroblasts derived from the same primary cells will have significantly greater aSMA levels (Yin et al., 2008). In addition to aSMA, we have demonstrated that myofibroblasts, but not fibroblasts, from all tissues examined to date, including cornea, lung, skin, heart, and liver, all contain a Cl
current (IClLPA) activated by the phospholipid growth factors lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) (Wang et al., 2002a,b; Yin and Watsky, 2005; Yin et al., 2008). This whole cell current appears to be identical to the volume-activated Cl
current (Wang et al., 2002a; Watsky, 1995a; Yin and Watsky, 2005). Interestingly, we find that corneal keratocytes, but not myofibroblasts, contain large delayed rectifying Kþ currents and Naþ currents that are capable of firing action potentials under current clamp conditions (Watsky and Rae, 1992). In the cornea, the phenotypic distinction between cells is a bit more involved, particularly between keratocytes and fibroblasts. Experimentally, keratocytes are maintained in culture by excluding serum from the culture medium, whereas addition of serum promotes the fibroblast phenotype (Beales et al., 1999). Compared to keratocytes, serum-derived corneal fibroblasts secrete less total glycosaminoglycan as keratan sulfate (Beales et al., 1999), possess no aldehyde dehydrogenase, and synthesize significantly higher levels of decorin and significantly lower levels of prostaglandin

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D synthase (Berryhill et al., 2002). Interestingly, it appears that fibroblasts can be restored to the keratocyte phenotype and that myofibroblasts can be returned to the fibroblast phenotype, but myofibroblasts cannot be returned to the keratocyte phenotype (Maltseva et al., 2001). Fibroblasts from most tissues appear to be coupled by gap junctions (Pitts et al., 1986). In some tissues, such as the skin, fibroblasts can be coupled to other cell types (Chilton et al., 2007; Kam et al., 1986; Miragoli et al., 2006, 2007; Zlochiver et al., 2008). In the heart, myofibroblasts appear to be coupled to myocytes, and this coupling has the potential to promote reentry-induced arrhythmias (Chilton et al., 2007; Miragoli et al., 2006, 2007; Zlochiver et al., 2008). We have demonstrated active intercellular coupling between keratocytes in the living rabbit cornea (Fig. 4.1) (Watsky, 1995b). Corneal fibroblasts and also myofibroblasts have been shown to be functionally coupled in culture (Spanakis et al., 1998), although it appears that this

A

C

7 min

B

1 min

D

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Figure 4.1 Dye spread through keratocyte gap junctions in a control rabbit cornea. (A) Bright field Hoffman modulation contrast image of the posterior-most keratocytes. (B–D) Fluorescent dye spread images of the same region captured at 1 min (10-s exposure), 7 min (20-s exposure), and 24 min (100-s exposure) after dye injection. Arrows point to the source cell. Open arrows point to cells within the same lamellar plane that are not coupled. Arrowheads point to coupled cells not residing within the same focal plane. Scale bars ¼ 100 mm. Reprinted from Watsky (1995b). Copyright by ARVO.

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Figure 4.2 Dye spread through keratocyte gap junctions in a rabbit cornea wounded 24 h earlier. (A) Bright field Hoffman modulation contrast image showing activated keratocytes migrating into the wound region (posterior cornea). (B) Fluorescent dye spread image of the same region captured 45 min (25-s exposure) after dye injection. Arrows point to the source cell. Arrowheads point to coupled cells not residing within the same focal plane. Scale bars ¼ 100 mm. Reprinted from Watsky (1995b). Copyright by ARVO.

coupling is diminished in the cornea during wound healing (Fig. 4.2) (Watsky, 1995b). We have postulated that electrical activity generated by keratocyte Kþ and Naþ current activity (possibly action potentials) could be carried across the cornea through the gap junction network (Watsky, 1995b; Watsky and Rae, 1992).

3. Myofibroblasts As described earlier, the defining property of myofibroblasts in all tissue types is the presence of aSMA. While in most instances myofibroblasts are derived from fibroblasts, they can also be derived from other diverse cell types, such as peripheral blood mononuclear cells (discussed in detail below) and even endothelial cells (Petroll et al., 1997, 1998). Myofibroblasts are considered the wound/insult-induced, activated phenotype of their precursor cell type. Under routine wound healing conditions, fibroblasts activate and differentiate into the myofibroblast phenotype, after which they migrate to the site of injury, remodel extracellular matrix at the site of injury, act to contract the wound via their aSMA activity, and then go through apoptosis. One of the primary causes of fibrotic pathologies is the failure of myofibroblasts to undergo apoptosis, allowing for continuous matrix production and resultant fibrosis (Desmoulie´re et al., 1995).

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In addition to aSMA and the IClLPA current described earlier, myofibroblasts have other distinct phenotypic characteristics. In the keratocyte to myofibroblast transition in the cornea, myofibroblasts lose the distinct Kþ and Naþ currents seen in the quiescent keratocytes (Watsky, 1995c). The ED-A splice variant of fibronectin and the Thy-1 cell surface glycoprotein have also been specifically associated with the myofibroblast phenotype (Bondi et al., 2009; Cohen et al., 2009; Koumas et al., 2003; Rajkumar et al., 2005).

4. Fibroblast to Myofibroblast Signaling 4.1. Transforming growth factor-beta (TGFb) The most common signaling pathway responsible for fibroblast to myofibroblast differentiation is the TGFb signaling pathway. TGFb receptors are divided into two subfamilies, type I and type II (TbR-I and TbR-II) (Luo and Lodish, 1996). TbR-I and TbR-II are similar proteins; both are glycoproteins with a single membrane-spanning segment and a serine– threonine protein kinase domain located in the C-terminal segment. A distinguishing feature of TbR-I is the GS domain, which becomes phosphorylated after TGFb binding and plays a critical role in TbR-I and TbR-II activation. TbR-I also contains a binding site for the FKBP12-binding protein, a negative regulator of receptor signaling. A crucial distinguishing feature of TbR-II is that it is constitutively active. TGFb binds to the TbR-II, which then alters its configuration to allow it to sequentially bind to TbR-I. The constitutively active TbR-II then phosphorylates the TbR-I GS domain following detachment of the inhibitory FKBP12-binding protein. At this point, the phosphorylated receptor complex is considered activated. TGFb signaling occurs through Smad proteins. Smad2 and/or Smad3 are bound to TbR-I in association with another protein called SARA. Following receptor phosphorylation and activation, Smad2/3 becomes phosphorylated, detaches from TbR-I and SARA, and moves into the cytoplasm where it associates with Smad4. This complex then migrates to the nucleus, where it binds to the Smad-binding element. Binding to the Smad-binding element and to additional transcription partners induces the cellular response (Gomperts et al., 2003). One transcription product induced by TGFb1 is Smad7, a negative regulator of TbR-I activation (Nakao et al., 1997). Smad7 associates with TbR-I and prevents access of the receptor to SARA and Smad2.

4.2. TGFb cooperative proteins and independent pathways Several proteins have been shown to interact with the TGFb signaling pathway (Hinz et al., 2007). Downstream members of the Wnt signaling pathway have been shown to directly interact with Smad4, leading to synergistic effects between TGFb and Wnt on Xenopus development (Nishita et al., 2000).

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Interactions between TGFb and Wnt signaling pathways have also been shown to control activation of specific target genes through Smads (Labbe et al., 2000, 2007; Letamendia et al., 2001). This TGFb/Wnt synergy has also been demonstrated to influence the effect of TGFb on chondrocyte and adipocyte differentiation (Zhou et al., 2004). Interactions between TGFb and Wnt signaling through beta-catenin have been shown to mediate transcription in human dermal fibroblasts via Smad3 and the p38 MAPK pathway, possibly playing a role in hypertrophic scarring (Sato, 2006). Possible interactions between TGFb and Wnt signaling pathways have also been implicated in pulmonary fibrosis (Liu et al., 2009; Salazar et al., 2009). Connective tissue growth factor has been shown to be induced by and to cooperate with TGFb, potentiating the profibrotic activities of the TGFb and also inducing fibroblast proliferation (Leask et al., 2002). Galectin-3, a cell surface protein, is required for differentiation of cultured hepatic stellate cells into myofibroblasts. Disruption of the galectin-3 gene blocks myofibroblast differentiation and procollagen (I) expression both in vitro and in vivo, significantly reducing hepatic fibrosis. Addition of exogenous galectin-3 restores differentiation and procollagen expression (Henderson et al., 2006). The TGFb signaling pathway has also been shown to interact with the Notch signaling pathway, an evolutionarily conserved pathway that determines cell fate by regulating numerous cell processes, including proliferation, differentiation, and apoptosis. This appears to be mediated through the Smad pathway (Blokzijl et al., 2003; Fu et al., 2009). Myofibroblast differentiation can also be inhibited by interfering with TGFb signaling. Increasing cytoplasmic cAMP has been shown to antagonize TGFb-mediated keratocyte to myofibroblast differentiation in the cornea. It appears that this inhibitory pathway is not working through Smads or the MAPK signaling pathway, but instead through the activation of RhoA (Xing and Bonanno, 2009). Fibroblast to myofibroblast differentiation has also been shown to progress through TGFb-independent pathways. LPA has recently been associated with fibrotic processes and diseases and may possibly work through this route, although some associations between LPA and TGFb have been reported (see below). We were the first to demonstrate that LPA can trigger fibroblast to myofibroblast differentiation (Yin and Watsky, 2005). As noted earlier, we have also described an LPA/S1P-activated Cl
current (IClLPA) that is active in myofibroblasts but not in myofibroblast precursors. Importantly, we also demonstrated that blocking IClLPA activity can prevent fibroblast to myofibroblast differentiation (Yin and Watsky, 2005; Yin et al., 2008). In addition, Rho-associated kinases, which are activated by LPA, have been found to stimulate myofibroblast differentiation from cultured human skin systemic sclerosis (SSc) fibroblasts. Rho-associated kinases were also found to stimulate the production of extracellular matrix by these cells (Akhmetshina et al., 2008). Clinically, LPA has been linked to several forms of fibrosis, including hepatic (Ikeda et al., 1998; Tangkijvanich et al., 2002; Watanabe et al., 2007), renal (Eberlein et al., 2001; Pradere et al., 2007,

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2008a), and pulmonary fibrosis (Ley and Zarbock, 2008; Tager et al., 2008; Yin et al., 2008). LPA has also been found to promote wound healing in the skin (Balazs et al., 2000, 2001; Demoyer et al., 2000), possibly through its ability to stimulate keratinocyte laminin 5 synthesis (Amano et al., 2004). Nerve growth factor (NGF) is another pro-myofibroblast protein. While NGF is best known for stimulating nerve growth, it also appears to play a role in tissue repair and inflammatory processes. It has been shown that NGF stimulates fibroblast to myofibroblast differentiation in both human lung and skin fibroblasts (Micera et al., 2001). Interestingly, while NGF induces aSMA expression and wound contraction in these cells, it does not influence collagen production or metalloproteinase production, physiological hallmarks of many myofibroblasts. It has not been determined whether possible TGFb autocrine production is involved in the NGFstimulated myofibroblast differentiation. Although its cooperative interactions with TGFb have not specifically been studied, platelet-derived growth factor (PDGF) also induces fibroblast to myofibroblast differentiation. PDGF-BB has been shown to induce renal tubulointerstitial fibroblast to myofibroblast differentiation (Tang et al., 1996), and PDGF-A induces myofibroblast differentiation of lung alveolar fibroblasts (Bostrom et al., 1996). More recently, PDGF-B has been implicated in corneal myofibroblast differentiation (Kaur et al., 2009). Interleukin (IL)-6 has also been suggested as a TGFb-independent stimulator of wound healing and myofibroblast differentiation (Gallucci et al., 2006), as has Fizz1 (found in inflammatory zone), a protein secreted by alveolar epithelial cells (Liu et al., 2004). In addition, in vitro fibroblast aSMA expression has been shown to be induced by thrombin (Bogatkevich et al., 2001), endothelin-I (Shi-Wen et al., 2004), and angiotensin-II (Swaney et al., 2005).

5. Mononuclear Cells in Peripheral Blood That Differentiate into Myofibroblasts 5.1. Historical perspective In 1882, Metchnikoff described the transformation of blood mononuclear cells into fibroblasts during cicatrization in the tail fin of tadpoles (Metchnikoff, 1882). In 1928, Maximov described fibroblasts growing from cultures of blood lymphocytes and monocytes (Maximow, 1928).

5.2. Neofibrosis This topic was revisited in 1991 when Labat et al. observed that blood monocytes from patients with osteomyelosclerosis and Engelmann’s disease (Labat et al., 1991b) and HLA-DRþ blood monocytes from patients with

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cystic fibrosis (Labat et al., 1991a) spontaneously developed, when placed in culture, into fibroblast-like cells that he termed ‘‘neofibroblasts.’’ Additional studies showed that these neofibroblasts were pluripotent, secreting not only type I collagen but also uromodulin, amyloid-b peptide, a-fetoprotein, and carcinoembryonic antigen (Bringuier et al., 1992).

5.3. Fibrocytes In 1994, Bucala et al. described a cell population isolated by culturing human peripheral blood mononuclear cells in low-serum-containing medium on fibronectin-coated surfaces. These cells, termed ‘‘fibrocytes,’’ expressed type I collagen (col1), CD11b, CD13, CD34 (a pan-hematopoietic stem cell marker), CD45-RO (common leukocyte antigen), major histocompatibility complex class II, and CD86 but were negative for aSMA, CD14, CD16, CD25, CD10, CD38, and CD19 (markers for monocytes, dendritic cells, and B cells) (Bucala et al., 1994). Studies using bone marrow chimeric mice have shown that tissue fibrocytes originate from the bone marrow (Mori et al., 2005). As fibrocytes are cultured longterm in vitro and in some in vivo inflammatory models, they lose CD34 and CD45 expression, and they differentiate into a myofibroblast phenotype when cultured with TGFb, endothelin, IL-4, IL-13, or PDGF-B (Oh et al., 1998; Strieter et al., 2009a, 2009b). There has been an expansion of knowledge about what markers are present on fibrocytes as detected by flow cytometry in the peripheral blood. Some consider circulating cells that stain CD45þ and col1 as fibrocytes because 100% of col I-staining cells are CD45þ, whereas only 10% of col Istaining cells are CD34þ (both are CD14
) (Strieter et al., 2009b). Some studies, however, suggest that fibrocytes may differentiate from CD14þ/ CD16/CD11bþ/CD64þ/CD32þ blood monocytes (Abe et al., 2001; Pilling et al., 2003, 2006; Varcoe et al., 2006; Yang et al., 2002), while others question whether fibrocytes are truly derived from CD14þ progenitor cells (Strieter et al., 2009a). The expression of certain chemokine receptors by fibrocytes is thought to be important in directing their migration to sites of tissue damage to initiate repair. Human fibrocytes have been reported to express CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4 (Bellini and Mattoli, 2007; Strieter et al., 2009a). The CXCR4/CCL12 axis has been demonstrated to be important in trafficking of fibrocytes to the lungs of mice with bleomycin-induced pulmonary fibrosis and in induction of chemotaxis of fibrocytes in vitro (Phillips et al., 2004). Fibrocytes have been implicated in contributing to the pathogenesis of several fibrotic conditions. In nephrogenic systemic fibrosis (induced in patients with renal function impairment who were given gadolinium-based contrast agents for gadolinium-enhanced magnetic resonance imaging), fibrocytes that are CD34þ/

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col Iþ/procol Iþ are found in the fibrotic skin lesions in increased numbers (Bucala et al., 1994). Patients with interstitial pulmonary fibrosis have been shown to have higher levels of peripheral blood monocytes and CXCR4þ/ CD45þ/col Iþ fibrocytes (Pierce et al., 2007), and small numbers of CD34þ/ procollagenþ or CD34þ/a SMAþ fibrocytes have been found in fibroblastic foci in lungs of patients with idiopathic pulmonary fibrosis (AnderssonSjoland et al., 2008). High levels of circulating fibrocytes have been shown to correlate with increased disease activity and early mortality in patients with idiopathic pulmonary fibrosis (Moeller et al., 2009). In neonatal hypoxia models of pulmonary remodeling and pulmonary arterial hypertension in rats and calves, fibrocytes have also been identified in perivascular mononuclear populations and are postulated to be essential for pulmonary adventitial remodeling (Frid et al., 2006). The airway remodeling characteristics of bronchial asthma are accompanied by accumulation of fibrocytes that may differentiate into myofibroblasts through the action of locally produced TGFb1, IL-4, and IL-13 (Bellini and Mattoli, 2007). There is also upregulation of the CCR7 ligand CCL19 by endothelial cells, smooth muscle cells, and mast cells, which may contribute to recruitment of fibrocytes in these patients during asthma attacks (Kaur et al., 2006). Murine wound healing models have demonstrated that fibrocytes infiltrate the wound site and may (in addition to synthesizing matrix molecules) facilitate angiogenesis by producing vascular endothelial growth factor, PDGF-A, macrophage colony-stimulating factor, hepatocyte growth factor, granulocyte-macrophage colony-stimulating factor, basic fibroblast growth factor, and connective tissue growth factor (Hartlapp et al., 2001). In the prototypical fibrosing autoimmune disease, SSc (scleroderma), CD34þ fibrocytes were not observed to be elevated in biopsies from involved fibrotic skin of patients with the disease (Aiba et al., 1994). However, it should be noted that fibrocytes downregulate CD34 as they assume myofibroblast morphology and that the majority of fibroblasts in lesional skin of patients with SSc are myofibroblasts (Kirk et al., 1995). On the other hand, examination of blood of patients with limited cutaneous SSc failed to show increased numbers of cells expressing cell surface markers for circulating fibrocyte progenitors (Russo et al., 2007). A recent case report found that myofibroblasts grown from sclerodermatous skin of a patient with chronic graft-versus-host disease were of recipient rather than donor origin (Goussetis et al., 2009). In addition to EMT as a source of fibroblasts/myofibroblasts in the unilateral ureteral obstruction model of renal fibrosis in mice, both CD45þ/Col1þ and CD34þ/ Col1þ fibrocytes have been found to be present, suggesting another source of cells contributing to fibrosis (Sakai et al., 2006). Fibrocytes respond in culture to toll-like receptor (TLR)2, TLR4, and TLR7 ligands, polyIC or viral stimulation by producing large amounts of IL-6, and to viral stimulation by producing type I interferon, suggesting that they may play a role in host defense against pathogens (Balmelli et al., 2007). In an ovine model of intimal hyperplasia affected by placing a carotid artery

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synthetic patch graft, autologous peripheral blood mononuclear cells labeled with carboxyfluorescein diacetate succinimidyl ester were found to develop in CD34þ/CD45þ vimentinþ/aSMAþ fibrocytes in intimal lesions. Fibrocytes were identified (immunostaining for CD34/procollagen and leukocyte-specific protein-1/procollagen) in the fibrous cap of 35 of 40 specimens of human atherosclerotic plaques, suggesting that they may contribute to plaque stabilization (Medbury et al., 2008). In a rat model of transplant vasculopathy, the degree of vasculopathy correlated with the frequency of fibrocytes in the graft lesions (Onuta et al., 2009). In addition, fibrocytes are increased in peripheral blood of burn patients and in hypertrophic scars resulting from third degree burns of the skin (Yang et al., 2002, 2005). Human fibrocytes produce MIP-1a, MIP-1b, MCP-1, IL-8, GRO, M-CSF-a, and IL-6 (Chesney et al., 1998). It has been suggested that since fibrocytes secrete the T cell chemoattractants MIP-1a, MIP-1b, and MCP-1, they would be able to modulate CD4þ T cell migration into inflammatory sites where they can participate in T cell activation and immune induction. Fibrocytes may contribute to the fibrosis associated with schistosomiasis (Chesney, 2007). Circulating fibrocytes are activated (i.e., express phosphorylation activation of the p44/42 and p38 MAP kinases and STAT3 and STAT5) in patients with early and established rheumatoid arthritis (RA) compared to control individuals (Galligan et al., 2009). In the murine model of RA induced by immunization of DBA/1 mice with type II collagen, increased numbers of peripheral blood fibrocytes expressing STAT5 were observed at early time points in the disease, and there were elevated numbers of fibrocytes in arthritic paws compared to paws of control mice (Galligan et al., 2009). Galligan et al. (2009) suggest that fibrocytes may contribute to the synovial fibroblast pool in patients with RA and may modulate the immune and inflammatory response in RA by secreting cytokines/chemokines. Fibrocytes can act as antigen-presenting cells via expression of major histocompatibility complex class II molecules and costimulatory molecules and have been shown to stimulate human tetanus toxoid responses and prime CD8 T cell responses (Balmelli et al., 2005; Chesney et al., 1997). Fibrocytes are able to differentiate into adipocytes, and this process is mediated by peroxisome proliferation-activated receptors (Hong et al., 2007). Several agents have been described that curtail fibrocyte formation, including serum amyloid P, aggregated IgG, and blockade of adenosine A2A receptors (Abe et al., 2001; Katebi et al., 2008; Pilling et al., 2003).

5.4. Other monocyte-derived progenitors of fibroblast-like cells There have been reports of other fibroblast-like cells derived in vitro from monocyte progenitors that share some characteristics of the expanded definition of fibrocytes (from Bucala’s original description;

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Bucala et al., 1994) that tend to have a great deal of plasticity with regard to their ability to differentiate into other cell types. The term ‘‘monocyte-derived mesenchymal progenitors’’ (MOMPs) was used by Kuwana et al. to describe fibroblast-like cells derived from CD14þ monocytes from normal humans. These spindle-shaped cells expressed CD14/CD45/CD34/Col1 and had an absolute requirement of growth on fibronectin-coated surfaces with low glucose and 10% fetal calf serum and also required exposure to CD14
peripheral blood mononuclear cells (Kuwana et al., 2003). When grown in the presence of specific growth factors or medium, MOMPs have the ability to differentiate into other mesenchymal cells, including osteoblasts, myocytes, adipocytes, and chondrocytes (Kuwana et al., 2003). MOMPs also express CD13, CD116, CD11c, CD64, HLA Class I, HLA-DR, CD40, CD86, CD29, CD44, CD54, CD105/SH2, CD31, CD144, Flt-1, Ac-LDL, Col3, fibronectin, and vimentin (Kuwana et al., 2003). CD45þ/CD14þ/CD35þ/Col1þ fibroblast-like cells called pluripotent stem cells or f-macrophages (described by Zhao et al.) are grown from CD14þ human mononuclear cells on chamber slides coated with collagen and in RPMI 1640 medium supplemented with 7% fetal calf serum, monocyte colony-stimulating factor, and leukemia inhibitory factor (Zhao and Mazzone, 2005; Zhao et al., 2003). These cells assume a spindle-shaped morphology after 7 days in culture and can be differentiated into epithelial cells, CD3þCD8þ T cells, neuronal cells, or hepatocytes when cultured in the appropriate medium and with specific growth factors (Zhao et al., 2003). These f-macrophages can be expanded by culturing then with low-dose thrombopoietin (Zhao and Mazzone, 2005).

6. Myofibroblast-Associated Pathologies 6.1. Myofibroblasts and cardiac repair following infarction Following myocardial infarction (MI), cardiac repair occurs at the infarcted myocardium, beginning with inflammatory and angiogenic responses, followed by fibrous tissue accumulation (scarring). Scar formation at the site of MI preserves cardiac structural integrity and is essential to the heart’s recovery. Cardiac repair is a highly regulated process. It begins with the activation of latent matrix metalloproteinases (MMPs), which degrade the existing extracellular matrix and coronary vasculature (Cleutjens et al., 1995a). This proteolytic activity declines by the end of week 1 post-MI and is coincident with the increased expression of MMP inhibitors, termed tissue inhibitors of MMPs or TIMPs (Sun et al., 2000). Circulating inflammatory cells that include neutrophils and monocytes/macrophages arrive at the infarct site

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soon after MI. They contribute to the proteolytic digestion and phagocytosis of infarcted tissue. These inflammatory cells home to the site of the MI, drawn by adhesion molecules and chemoattractant cytokines (or chemokines) expressed by the endothelial cells of the coronary vasculature that borders on the infarct site. Their migration into the infarct site is facilitated by MMP proteolytic activity. Inflammatory cells release proinflammatory/ profibrotic cytokines and growth factors to facilitate cardiac repair. This inflammatory response peaks at weeks 1 and 2 post-MI and then tapers off as inflammatory cells disappear from the infarct site within 3–4 weeks following MI, a consequence of their programmed cell death. The fibrogenic component, which substitutes for lost parenchymal cells, follows the initial phase of collagen degradation. It begins with the activation of TGFb1, the key mediator of fibrogenesis. Increased synthesis of fibrillar type III and type I collagens is preceded by an increased expression of their mRNA transcripts (Cleutjens et al., 1995b). Collagen fibers are morphologically evident at week 1 post-MI, while an organized assembly of these fibers in the form of scar tissue becomes evident at week 2 (Sun et al., 1994). This assembly continues to accumulate over a period of 8 weeks. 6.1.1. Cells responsible for fibrous tissue formation in the infarcted myocardium Cells responsible for fibrous tissue formation at the site of MI consist principally of phenotypically transformed fibroblast-like cells having distinctive morphologic features and phenotypic characteristics. These cells express aSMA microfilaments that give them contractility, and are thus considered myofibroblasts (Desmoulie´re and Gabbiani, 1996; Gabbiani et al., 1971). Following MI, myofibroblasts first appear at the border zone between the infarcted and noninfarcted myocardium soon after the arrival of inflammatory cells, then appear within the infarct site. Cells that account for the appearance of myofibroblasts remain to be fully elucidated, but resident fibroblasts located at the border zone are primary candidates (Yano et al., 2005). Monocyte-derived macrophages in the infarcted myocardium are also reported to be able to differentiate into myofibroblasts (Fujita et al., 2007). Circulating precursor cells of bone marrow origin may contribute to the appearance of myofibroblasts in the infarcted myocardium, but results are contradictory. van Amerongen et al. (2008) reported that up to 24% of myofibroblasts in the infarcted myocardium are bone marrowderived cells at day 7 post-MI, while Yano et al. (2005) have reported that bone marrow-derived cells are not the origin of myofibroblasts in the infarcted myocardium. In addition, it has been reported that pericytes also serve as a source of myofibroblasts in obstructive fibrosis of the kidney (Lin et al., 2008). Thus, myofibroblasts that appear in the infarcted myocardium could be derived from various origins. Specific factors that facilitate myofibroblasts differentiation process have been identified. It is presumed

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that TGFb1, elaborated by macrophages, governs the appearance of the myofibroblast phenotype (Desmoulie´re et al., 1993). Evidence supporting this hypothesis can be seen in cases of ischemia–reperfusion, wherein TGFb1 plays a central role in the oxygen-dependent differentiation of cardiac fibroblasts to myofibroblasts triggered by perceived hyperoxia (Roy et al., 2003). It is also reported that alpha(3) integrin interacts with type VI collagen to promote myofibroblast differentiation (Bryant et al., 2009). After appearance, myofibroblasts rapidly proliferate and accumulate in the infarcted myocardium, peak at weeks 1 and 2, and then gradually decline when healing is completed. 6.1.2. The functions of myofibroblasts in the infarcted myocardium Myofibroblasts have a diverse portfolio of metabolic activities. First, myofibroblasts are responsible for formation of the fibrous tissue (scar) via their expression of type I and III fibrillar collagens at the mRNA and protein levels (Cleutjens et al., 1995b; Sun et al., 1998). Our studies have shown that type I collagen gene expression is significantly increased as early as day 3 post-MI and lasts for weeks. The increase in type I collagen mRNA is temporally and spatially coincident with accumulated myofibroblasts in the infarcted heart (Fig. 4.1). Using immunohistochemistry, our study shows that myofibroblasts are colocalized with accumulated collagen (Fig. 4.3), further indicating the important role of myofibroblasts in collagen synthesis during cardiac repair. Second, myofibroblasts are an important source of proinflammatory cytokines, including tumor necrosis factor (TNF)-a, IL-1b, and IL-6 (Turner et al., 2009). Cultured cardiac myofibroblasts secrete these cytokines in response to specific stimuli, including hypoxia, mechanical stretch, and increased levels of cytokines, such as IL-1a (Turner et al., 2009). Thus, in addition to inflammatory cells, myofibroblasts also release proinflammatory cytokines and contribute to the inflammatory response in the infarcted myocardium. Myofibroblasts have also been shown to express natriuretic peptides, indicating the potential influence of these peptides on reparative fibrosis (Calderone et al., 2006). Third, studies have shown that myofibroblasts express renin, angiotensinconverting enzyme (ACE), and type I angiotensin (Ang) receptors at the infarct site (Fig. 4.4) (Passier et al., 1996; Sun and Weber, 1996a,b,c; Sun et al., 2001). Furthermore, myofibroblasts obtained from the 4-week-old infarct scar and studied in culture under serum-deprived conditions that eliminate circulating renin, ACE, and Ang I are found to express angiotensinogen, cathepsin D, ACE, and Ang II receptors (Katwa et al., 1997). These findings indicate that locally produced Ang II may regulate the function of myofibroblasts in an autocrine manner. Given the presence of aSMA in myofibroblasts, which is also present in vascular smooth muscle cells, it is reported that Ang II, endothelin-1, and vasopressin promote scar tissue

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Figure 4.3 Type I collagen gene expression and the colocalization of myofibroblasts and collagen in the infarcted myocardium. Normal heart expresses a low level of type I collagen mRNA (panel A). At day 7 post-MI, type I collagen mRNA is largely increased at the site of MI (panel B). Immunohistochemical aSMA detection reveals accumulated myofibroblasts (mf) at the infarcted myocardium at day 7 post-MI (panel C), which are colocalized with accumulated collagen (panel D, red). S, septum; RV, right ventricle; N, necrotic tissue. Panels C and D: 200. Reprinted with publisher’s permission from Sun et al. (2000).

contraction (Goto et al., 1998; Katwa et al., 1997). Gabbiani et al. (1971) have demonstrated the contractile behavior of myofibroblasts in scar tissue. Myofibroblasts and their aSMA microfilaments are joined to one another through gap junctions. This creates a contractile scar tissue assembly. Their contractile properties may help to prevent the infarct area from dilatation (Blankesteijn et al., 1997). Ang II has also been shown to promote collagen synthesis by myofibroblasts via stimulating TGFb (Sun et al., 1998).

6.2. Systemic sclerosis SSc, also known as scleroderma, is an often fatal disease characterized by progressive, widespread tissue fibrosis. Early diagnosis of SSc is difficult, and the only treatments available are for symptomatic pathologies, with no

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Figure 4.4 Renin, ACE, and Ang II receptor expression in the infarcted myocardium. By in situ hybridization, a high level of renin mRNA is expressed at the site of MI, but not in noninfarcted myocardium (nonMI) (panel A). Detected by autoradiography, high binding density of ACE (panel C) is observed at the site of MI. Immunohistochemistry reveals that myofibroblasts are responsible for the expression of renin (panel B, brown) and ACE (panel D, arrowheads). Panel E shows abundant myofibroblasts (brown) in the center of infarcted myocardium at week 4 post-MI. High Ang II receptor binding (black grains) is seen in myofibroblasts, whereas blood vessels (V1, V2) and fibroblasts (Fb) express low density of Ang II receptors. Panels A and B reprinted with publisher’s permission from Sun et al. (2001). Panels C and D reprinted with publisher’s permission from Sun et al. (1994). Panels E and F reprinted with publisher’s permission from Sun and Weber (1996c).

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treatments available for the underlying disease (Steen, 2006). SSc has no known cause, although it does have an autoimmune component. SScassociated fibrosis is similar to that seen during wound healing, only the process is not self-limiting as in wound healing ( Jun et al., 2003). SSc and other fibrotic diseases, including fibrotic wound healing, are characterized by excessive extracellular matrix deposition and soft tissue deformation. This excessive matrix deposition is likely the result of a dynamic process involving increased synthesis of type I and III collagen and other matrix proteins, coupled with a decrease in metalloproteinase production and an increase in the production of tissue inhibitors of metalloproteinases (Arthur, 2000). Clinically, SSc-associated fibrosis may involve multiple tissues. Fibrosis of the skin and pulmonary system are particularly common, and this may manifest as skin tightening and dyspnea. Modified Rodnan skin scores (MRSS) are a validated and widely used method of measuring the extent and severity of skin thickening in SSc (Clements et al., 1990; Merkel et al., 2003). Pulmonary function tests, including forced vital capacity (FVC) and diffusion capacity of carbon monoxide (DLCO), are used to detect (and monitor) lung involvement in SSc (Beretta et al., 2007). Pulmonary hypertension is a leading cause of death in SSc. Bioactive lysophospholipids are a relatively recently described class of lipids that signal through both cell membrane and intracellular receptors. LPA and S1P are two of the major bioactive lysophospholipids. LPA has several synthesis pathways; one of the primary pathways is from lysophosphatidyl choline by the action of lysophospholipase D, also known as autotaxin. Activated platelets, a hallmark of SSc, are a major source of LPA, and many other cell types have also been found capable of synthesizing LPA (Aoki, 2004). The biological activities of LPA include mitogenic effects, antimitogenic effects, regulation of Ca2þ homeostasis, regulation of the actin cytoskeleton, and inhibition of apoptosis (Goetzl and An, 1998; Tigyi et al., 1999). In addition, LPA affects migration in a number of cell types (Takuwa et al., 2002). S1P is generated from sphingosine by sphingosine kinase. Sphingosine precursors include sphingomyelin and ceramide. S1P has specific immune system effects (see below) and plays a role in cell growth and survival, angiogenesis, and neural growth. Work from our laboratory has demonstrated that LPA and S1P levels are elevated in SSc plasma (Tokumura et al., 2009) and appear to be involved in SSc myofibroblast differentiation (unpublished data). To date, 13 mammalian receptors have been cloned that are specifically activated by lysophospholipids. Eight of these receptors are recognized as extracellular LPA receptors (designated LPA1–LPA8), while five are S1P receptors (designated S1P1–S1P5) (Meyer zu Heringdorf and Jakobs, 2007; Rivera and Chun, 2008; Tabata et al., 2007). LPA also acts as an agonist toward the PPARg receptor (McIntyre et al., 2003). In all cell types studied to date, the signaling pathway for cell membrane-based LPA

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receptor-induced responses has been shown to be G-protein-mediated, with each receptor subtype capable of coupling to multiple G-proteins (Rivera and Chun, 2008). LPA1 has recently been associated with both renal and pulmonary fibrosis (Pradere et al., 2008; Tager et al., 2008). In the bleomycin-induced lung fibrosis animal model, Tager et al. (2008) found that absence of the LPA1 receptors impairs recruitment of fibroblasts into fibrotic lesions. Regarding skin, LPA1 was found to be upregulated in blistered skin, and LPA levels were elevated in blister fluid (MazereeuwHautier et al., 2005). LPA-mediated calcium signaling in keratinocytes seems to be mediated through LPA2 (Lichte et al., 2008). LPA has also been shown to increase keratinocyte proliferation and migration and also skin fibroblast proliferation, likely through a Gai (PTX-sensitive) signaling pathway (Piazza et al., 1995; Pietruck et al., 1997; Sauer et al., 2004). LPA has further been shown to activate Smad3 in keratinocytes (Sauer et al., 2004); Smad3 is a downstream signaling protein associated with TGFb signaling (see below). More recently, Xu et al. (2009) discovered that LPA induces avb6 integrin-mediated TGFb activation in epithelial cells via the LPA2 receptor. TGFb has also been associated with the pathobiology of SSc (Allanore et al., 2007; Ihn, 2008; Prud’homme, 2007). A few studies have examined the interactions of LPA and S1P, and TGFb. Sato et al. (2004) found that LPA inhibited TGFb-mediated collagen production in foreskin fibroblasts. Their data indicated that this response was due to LPA activation of ERK, which then destabilized the collagen mRNA. In a study looking at keratinocytes, Sauer et al. (2004) found that LPA activated Smad3 without altering TGFb release. On the other hand, in a recent Langerhans cell study looking at S1P, it was found that S1P could activate Smads and induce cell migration, potentially through interactions with TGFb. In this same study, LPA was found to be ineffective at inducing cell migration (Radeke et al., 2005). S1P has also been found to increase aSMA expression in lung fibroblasts (Urata et al., 2005). In a limited number of studies, TGFb signaling has been studied in SSc skin cells. Dong and colleagues found deficient Smad7 expression in SSc skin fibroblasts. This was linked to an increase in TGFb-stimulated Smad2,3 phosphorylation in these cells (Dong et al., 2002). Conversely, Asano et al. (2004) found increased Smad7 levels in SSc fibroblasts compared to controls, but impaired Smad7–Smurf interactions. Along different lines, it has also been shown that an increased TbRI:TbRII ratio may underlie aberrant TGFb signaling in SSc fibroblasts and contribute to elevated basal collagen production (Pannu et al., 2004). The most widely used animal model for SSc is the tight-skin mouse (TSK/þ) (Pablos et al., 2004). This mouse has a fibrillin-1 mutation and immunological abnormalities that lead to cutaneous fibrosis, increased lung collagen content, and enlarged air spaces similar to those seen in emphysema, and, with advanced age, progressive myocardial fibrosis and

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hypertrophy (Green et al., 1976; Osborn et al., 1987; Szapiel et al., 1981). In a study by Matsushita et al. (2007), TSK/þ mice were crossed with intracellular adhesion molecule-1 (ICAM-1) and L-selectin knockout mice to generate TSK/þ mice lacking these adhesion molecules. It was determined that a deficiency of ICAM-1, but not L-selectin, significantly suppressed the development of dermal sclerosis in these mice.

7. Summary Fibroblast to myofibroblast differentiation is critical for normal wound healing to occur, but when unchecked can lead to life-threatening pathological conditions. The most studied signaling pathway leading to fibroblast to myofibroblast differentiation is the TGFb pathway, which traditionally signals through Smad proteins. This review discusses this pathway, along with several ‘‘nontraditional’’ pathways, leading to myofibroblast transformation, and not always from fibroblasts. It is well established that the bone marrow-derived mononuclear cells enter the circulation and can morph into fibroblast-like cells/fibroblasts/myofibroblasts in vitro and in disease states in vivo. Reports suggest that there may be more than one circulating progenitor cell population that gives rise to fibroblasts/myofibroblasts, and such a redundant system may have survival advantage. The relationship of the various progenitors to each other has not been yet defined. SSc and MI are two pathologies in which myofibroblasts play significant roles. The fibrosis associated with SSc is directly related to myofibroblast activity. In SSc, as in all instances of fibroblast to myofibroblast differentiation we have examined, changes in ion channel activity appear to be required for the differentiation of fibroblasts into myofibroblasts. It is likely that circulating cells also contribute to the myofibroblast pool seen in fibrotic SSc tissues and organs. SSc-associated fibrosis appears to be only detrimental to patients suffering from this disease; myofibroblast-mediated fibrosis following MI actually appears to play a positive role by preserving cardiac structural integrity. Following MI, myofibroblasts appear and accumulate in the infarcted myocardium. As in SSc, myofibroblasts found in MI scar tissue are likely derived from several types of cells, including residential fibroblasts, macrophages, and possibly bone marrow-derived progenitor cells. Myofibroblasts have a diverse portfolio of metabolic activities. They play a major role in fibrous tissue formation at the infarct site. In addition, myofibroblasts secrete proinflammatory cytokines, thus contributing to the cardiac inflammatory response. Furthermore, myofibroblast-derived ACE produces Ang II, which may regulate the function and behavior of myofibroblasts in an autocrine manner.

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Index

A Actin cytoskeleton, 150–151 Alpha smooth muscle actin (aSMA), 167 Angiotensinconverting enzyme (ACE), 178, 180 Angiotensin (Ang) receptors, 178, 180 B Birds, meiotic silencing, 121 Blood mononuclear cells transformation, 172–176 C Caenorhabditis elegans, meiotic silencing in animals, 92–93 chromatin regulation, in germ line, 93–95 function of, 125 germ line development, 113 chromosome evolution, 117 DNA insertions, repression of, 114 embryogenesis, epigenetic control of, 115–116 genome integrity maintenance, 115 X chromosome, transcriptional regulation, 116–117 mechanism of, 124–125 mechanistic and functional comparison of in birds, 121 in insects, 118–119 in mammals, 119–121 N. crassa, 118 in vertebrates, 121–122 noncoding RNA and chromatin structure, 122–124 repressive mechanisms, in germ line dynamic pattern of, 98 extrachromosomal transgenic arrays, 97–98 genome-wide analysis of, 100–101 histone modifications in, 96–97, 99 H3K9me2 enrichment on male X, 101–103 H3K27me3, enrichment on X chromosome, 109–110 H3K9me2, regulators of, 104, 107–109 H3K9me2, siRNA-mediated pathway, 103, 105–106 inferred pairing and synapsis, 102 MET-2 in H3K9, 103 organization of, 95–96

polycomb repressive complex 2 (PRC2) system, 110–111 SiRNA functional pathways, 106–107 X chromosome, 96, 99–100 Cardiac repair, myofibroblasts cells for, 177–178 collagen synthesis, 178–179 matrix metalloproteinases (MMPs), 176–177 renin, ACE, and Ang II receptor expression, 178, 180 Caveolae Cav1 regulation of, 142–144 scaffolding domain, 143–145 and caveolin proteins isoforms, 140–141 oligomerization, 141–142 topology, 140 raft-dependent endocytosis dynamin, 146–147 GFP, 147 intersectin, 146 SV40, 146–147 Cholesterol role, raft-dependent endocytosis, 150 Chromatin dynamics, perichromatin region, 25–26 Chromatin loops, nuclear architecture cryoelectron microscopy, vitrified sections of, 14–15 3D-FISH of, 16–17 heterochromatin and euchromatin, 15–16 Chromosome conformation capture (3C), 6–7 Chromosome evolution, C. elegans germ line, 117 Chromosome territories (CTs) and chromatin domains mobility of, 20–21 nonrandom arrangements of, 18–20 history of, 7 and interchromatin spaces 3D reconstruction of, 12–13 ICN model, 14 laser–UV-microirradiation experiments, 8 Collagen synthesis, myofibroblasts, 178–179 4Pi Confocal laser scanning microscopy holographic focusing, 53–54 principle, 52 Confocal laser scanning microscopy (CLSM), 47 Correlation microscopy, nuclear architecture, 61–63

193

194

Index D

Detergent-resistant membranes (DRMs), 136–137 DNA insertions, C. elegans germ line, 114 DNA repair, nuclear topography double strand break (DSB) repair, 33 g-H2AX accumulations, ion microbeam irradiation, 32 laser and ion microbeams, 31 microirradiated chromatin, 31–32 perichromatin region (PR), 30 DNA replication, 29–30 E Electron microscopy (EM), 44–46 Embryogenesis, epigenetic control, 115–116 Endocytosis, raft-dependent actin cytoskeleton, 150–151 caveolae, 146–147 noncaveolar dynamin-dependent pathway, 147–148 noncaveolar dynamin-independent endocytosis, 148–149 pathways, 144 regulation of Cav1, negative regulation, 149–150 cholesterol role, 150 signaling cascades modulation, 151–152 Epidermal growth factor receptors (EGFR), 151–152 Epigenetics chromatin language, 2 and epigenomics, 3 Epigenomics, 3 Extrachromosomal transgenic arrays, 97–98 F Far-field fluorescence microscopy Abbe/Rayleigh limit, 50–52 conventional resolution background fluorescence, 50 CLSM, 47 3D FISH, 47–48 limitations of, 48–49 splicing speckle, 49 light optical nanoscopy, 51 4Pi confocal laser scanning microscopy holographic focusing, 53–54 principle, 52 SMIM, 55, 57 SPDM conventional epifluorescence and nanoimaging, 59 photobleaching, 59–60 single molecule microscopy, 58 spectral signatures, 57–58 STED, 54

structured illumination microscopy (SIM), 54–56 in vivo imaging, nanometer scale, 60–61 Fibroblasts and intermediates corneal keratocytes, 167–168 intercellular coupling, 168 morphology, 167 wound healing, 169 to myofibroblast signaling, TGFb role galectin-3, 171 lysophosphatidic acid (LPA), 171–172 nerve growth factor (NGF), 172 platelet-derived growth factor (PDGF), 172 Smad proteins, 170 TbR-I and TbR-II, 170 Wnt signaling pathway, 170–171 Fibrocytes, blood mononuclear cells transformation chemokine receptors, 173–174 markers, 173 rheumatoid arthritis (RA), 175 scleroderma (SSc), 174 Fluorescence in situ hybridization (FISH), 16–17, 47–48 G Germ line, C. elegans meiotic silencing. See also Caenorhabditis elegans, meiotic silencing chromatin regulation, 93–95 chromosome evolution, 117 DNA insertions, repression of, 114 embryogenesis, epigenetic control of, 115–116 genome integrity maintenance, 115 X chromosome, transcriptional regulation, 116–117 H Histone modifications, C. elegans germ line, 96–97, 99 H3K9me2, meiotic silencing enrichment on male X, 101–103 germ line regulators of, 104 CHK-2 and SIN-3, 108–109 RHA-1 and HIM-17, 108 siRNA-mediated pathway csr-1, 105–106 EGO-1, 105 ekl-1 and drh-3, 105–106 RRF-3, 106 H3K27me3, meiotic silencing, 109–110 I Infarcted myocardium. See Myocardial infarction (MI) Insects, meiotic silencing, 118–119

195

Index

Interchromatin compartment (IC), 10 Interchromatin spaces and chromosome territories (CTs) 3D reconstruction of, 12–13 ICN model, 14 L Light optical nanoscopy, 51 Lipid raft definition, 137–139 endocytosis actin cytoskeleton, 150–151 caveolae, 146–147 noncaveolar dynamin-dependent pathway, 147–148 noncaveolar dynamin-independent endocytosis, 148–149 pathways, 144 regulation of, 149–150 signaling cascades modulation, 151–152 origin of, 136–137 Lysophosphatidic acid (LPA), 171–172, 181–182 M Mammals, meiotic silencing, 119–121 Meiotic sex chromosome inactivation (MSCI), 121 Meiotic silencing. See Caenorhabditis elegans, meiotic silencing Microscopic analysis, nuclear architecture correlation microscopy, 61–63 electron microscopy, 44–46 far-field fluorescence microscopy (see Far-field fluorescence microscopy) Monocyte-derived mesenchymal progenitors (MOMPs), 176 mRNA export, nuclear architecture, 27, 29 Myocardial infarction (MI), 177–180 Myofibroblasts blood mononuclear cells transformation fibrocytes, 173–175 f-macrophages, 176 history, 172 MOMPs, 176 neofibrosis, 172–173 cardiac repair and cells for, 177–178 collagen synthesis, 178–179 matrix metalloproteinases (MMPs), 176–177 renin, ACE, and Ang II receptor expression, 178, 180 phenotypic characteristics, 169–170 systemic sclerosis, 179, 181–183 N Neofibrosis, 172–173 Nerve growth factor (NGF), 172

Neurospora crassa, meiotic silencing, 118 Noncoding RNA (ncRNA) and chromatin structure, 122–124 Nuclear architecture, microscopic studies biophysical properties of, 42 chromatin loops cryoelectron microscopy, vitrified sections of, 14–15 3D-FISH of, 16–17 heterochromatin and euchromatin, 15–16 chromosome conformation capture (3C), 6–7 chromosome territories (CTs) history of, 7 laser–UV-microirradiation experiments, 8 correlation microscopy for, 61–63 electron microscopy (EM) cytochemical studies, 46 postembedding protocol, 45–46 preembedding protocol, 45 experimental approaches, 43 far-field fluorescence microscopy Abbe/Rayleigh theory, 50–52 conventional resolution, 46–50 4Pi confocal laser scanning microscopy, 52–54 SIM, 54–56 SMIM, 55, 57 SPDM, 55–60 STED, 54 in vivo imaging, nanometer scale, 60–61 forces for, 40–42 genes kissing, 23–24 nuclear convergence of, 23 repositioning, 22 genome-wide maps of, 6 higher order chromatin arrangements, 33–34 models for chromatin organization and topology of, 9 chromatin polymer models, 11–12 CT–IC, 10 giant loop field (GLF), 11 interchromatin network (ICN), 11 mRNA export, routes of, 27, 29 nonrandom arrangements, 21–22 TFF1/GREB1 loci, 24 topography and DNA repair, 30–33 DNA replication, 29–30 genes, decondensation of, 38 recondensation process, 38–40 transcription factories (TFs), 37 transcription organization models, 35 transcription chromatin dynamics, perichromatin region, 25–26 topography of, 26–28

196

Index P

Perichromatin region (PR), chromatin dynamics, 25–26 Platelet-derived growth factor (PDGF), 172 Polycomb repressive complex 2 (PRC2) system HIS-24 activity, 111 MES proteins, 110–111 R Raft domains. See Lipid raft Renin, 178, 180 S Scleroderma (SSc). See Systemic sclerosis Small-interfering RNAs (siRNAs), C. elegans functional pathways, 106–107 H3K9me2, 103, 105–106 Spatially modulated illumination microscopy (SMIM), 55, 57 Spectrally assigned localization microscopy (SALM), 55, 58, 61 Spectral precision distance/position determination microscopy (SPDM) conventional epifluorescence and nanoimaging, 59 photobleaching, 59–60 single molecule microscopy, 58 spectral signatures, 57–58 Stimulated emission depletion (STED), 54

Structured illumination microscopy (SIM), 54–56 Systemic sclerosis, 179, 181–183 T Topography, nuclear architecture DNA repair, 30–33 DNA replication, 29–30 genes, decondensation of, 38 recondensation process, 38–40 transcription factories (TFs), 37 transcription organization models, 35 Transcription chromatin dynamics, perichromatin region, 25–26 topography of, 26–28 Transcription factories (TF), 26–28 Transforming growth factor-beta (TGFb), myofibroblast signaling cooperative proteins and independent pathways, 170–172 Smad proteins, 170 TbR-I and TbR-II, 170 V Vertebrates, meiotic silencing, 121–122 X X chromosome silencing, C.elegans, 99–100, 116–117

C

A Nuclear envelope

1 Mb chromatin domains

Chromosome territory

Splicing speckle

Interchromatin compartment (IC)

Nucleolus

Perichromatin region

1 mm − Transcription CTs with IC invaginations

+ Transcription

Nuclear neighborhood Nuclear neighborhood for gene silencing for gene expression

D

B Chromosome territory

Gene A Splicing speckle

Gene B

5

Gene C

Nucleolus

Nuclear envelope

Cell changes in response to signals

Movement to different nuclear neighborhoods: genes A and C become highly active and gene B becomes silenced in heterochromatin

Jacques Rouquette et al., Figure 1.2 Different models of chromatin organization and topology of gene expression. (A) The chromosome territory–interchromatin compartment (CT–IC) model (Cremer and Cremer, 2001, 2006b; Cremer et al., 2000) argues for the coexistence of highly folded CTs built up from chromatin domains and a nearly DNA-free IC expanding between these domains. The perichromatin region contains decondensed chromatin and provides the border zone between the rather compact interior of chromatin domains and the IC (Fakan, 2004). The PR is the major functional, nuclear subcompartment. DNA transcription, RNA splicing, as well as DNA replication and DNA

tel 0 Mb

11 p15.5 1 Mb

2 Mb

p15.4 2.8 Mb

cen

13 BACs (91 genes)

Jacques Rouquette et al., Figure 1.5 3D-FISH of 15 BACs spanning a gene-dense region on 11p15.5 (cf. Fig. 1.2C). Human fibroblast nucleus (stained with TOPRO-3; false colored in gray) after multicolor 3D FISH of the two HSA 11 territories (green) together with a gene-dense 2.35 Mb region from HSA 11p15.5. Fifteen BACs were used for the delineation of this region in false colors: red for the most telomeric, blue for the most centromeric BAC, and yellow for 13 BACs covering the intermediate region as a contig except for 350 kb in the middle (not shown). The nuclear shape is represented by a maximum Z projection of TOPRO-3 sections. The 3D reconstruction of the gene-dense region reveals a finger-like chromatin protrusion with a compaction factor of 1:300 expanding from CT 11 (cf. Fig. 1 in Albiez et al., 2006).

repair take place within the PR (cf. Figs. 1.4 and 1.8). (B) The interchromatin network (ICN) model (Branco and Pombo, 2006; reproduced with permission) proposes that euchromatin is made up from chromatin fibers, which intermingle more or less homogeneously by constrained diffusion both in the interior of CTs and between neighboring CTs. Blue dots signify interchromosomal contacts maintained by tethering. (C) The giant loop field (GLF) model (Chubb and Bickmore, 2003) suggests that transcription occurs on giant chromatin loops which expand from the surface of CTs and form a field of intermingling loops. When transcription ceases, the giant loops collapse back onto condensed core domain of CTs, which is typically visualized by chromosome painting. (D) The long-range interaction (LRI) model (Figure 4-66/page 241; Alberts et al., 2008, reproduced with permission) shows the most extreme version of a giant loop model. Giant loops can be very long and expand throughout the whole nuclear space in order to carry genes located on them even to very remote nuclear sites. In this way several genes can congress within the same ‘‘nuclear neighborhood for gene expression’’ or expression hub (Kosak and Groudine, 2004) and be transcribed there in a coregulated manner. In addition, the LRI model suggests genes on very long giant loops can reach distant nuclear neighborhoods for gene silencing.

A Interchromatin Chromatin domains compartment

E Nuclear envelope

Speckle

B

F IC

C

G

D

H

Jacques Rouquette et al., Figure 1.8 Schematic drawing comparing two models of transcription organization within the in situ nuclear landscape. (A) A scheme of a partial nuclear section according to the CT–IC model (cf. Fig. 1.2A) with  1 Mb chromatin domains and clusters of several such domains, as well as the interchromatin compartment expanding between them with splicing speckles and nuclear bodies. An enlargement of the boxed insert is shown in (B) and (C). (B–D) According to the CT–IC model, major transcriptional activity is expressed in the PR occurring on the border of chromatin domains (roughly delimited by the red dotted line). In the case where the chromatin domains (in gray) are close to each other (within a distance of about 400 nm or less), Brownian chromatin movement may shortly put transcription sites together (B and D, blue dotted line) and then separate them again (C). (E) This partial nuclear section differs from the situation presented in (A) by the assumption that the interchromatin compartment in addition to splicing speckles and nuclear bodies contains numerous chromatin loops expanding from higher order chromatin domains, as well as transcription factories. An enlargement of the boxed insert is shown in (F) and (G). Note that a perichromatin region (PR) does not exist according to this scheme. Even the demarcation between higher order chromatin domains and the interchromatin compartment (cf. Fig. 1.3; Rouquette et al., 2009) may become abolished in case of more extensive outlooping of chromatin. Comparative studies of chromatin topographies with ultrastructural resolution

5 Phases for each z-section

3 image stacks at three angles

60⬚

60⬚

Jacques Rouquette et al., Figure 1.9 Structured illumination microscopy (SIM) in three dimensions. Top: Scheme of 3D-SIM: by using a diffraction grating, an illuminating sine wave pattern is produced in the object space. At each axial (z) position, five different phases of the sine wave pattern are recorded. Three image stacks are registered with the diffraction grating sequentially recorded into three positions 60 apart. This allows to calculate a 3D-SIM image with enhanced details. Bottom: Comparison of a deconvolved light optical nuclear midsection obtained with a conventional laser confocal scanning microscope (left) and a light optical midsection obtained with 3D-SIM (right). Simultaneous imaging of DNA, nuclear lamina, and nuclear pore complex (NPC) epitopes was performed in C2C12 cells following labeling with antibodies against lamin B (green) and NPC epitopes (red). DNA (blue) was counterstained with DAPI. Enlargements of the periphery of the whole 3D SIM nuclear image suggest channels starting at nuclear pores and permeating through the lamina and between chromatin clusters (cf. Fig. 1.4). These details are not recognizable in the confocal midsection. From Schermelleh et al. (2008), reprinted with permission from AAAS.

are necessary in nuclei from different species and cell types with high and low global transcription rates in order to test whether transitions states between the nuclear topographies shown in (A) and (E) exist. (F–H) Transcription factories (TFs) (blue) are located in the interchromatin space expanding between chromatin clusters (cf. Fig. 1.6A). Several large chromatin loops containing active genes pass through TFs (cf. Fig. 1.6A). Locally constrained movements of chromatin clusters do not basically change the topolography of the loops simultaneously recruited by a given TF. IC: interchromatin compartment; small blue dots: RNA polymerase II; gray loops: DNA containing transcribed sequences emerging from chromatin domains; short green filaments: perichromatin fibrils (RNA transcripts) loaded with hnRNP proteins illustrated in red; black dotted line circles indicate places of partial chromatin decondensation taking place in the PR. Bars: (A, E) 500 nm; (B, C, F, G) 200 nm; (D, H) 60 nm.

A

C

B

D

2 mm

E

500 nm

100 nm

Jacques Rouquette et al., Figure 1.11 Comparison of conventional epifluorescence microscopy and nanoimaging of mRFP-tagged histones H2A (red) and GFP-tagged Snf2H transcription factors (green) within a human U2OS nucleus (Gunkel et al., 2009). (A) Conventional epifluorescence image. (B) 2CLM image, here the position of individual mRFP-tagged H2A histones and GFP-tagged Snf2H transcription factors is visualized as a single dot with a size representing the individual localization accuracy. (C)–(E) display Enlargements from the boxed regions in (A) and (B), respectively. Note that the magnification of the conventional image is empty, that is, it does not provide better resolved structural details in contrast to (D) and (E). Scale bars are 2 mm in (A) and (B), 500 nm in (C) and (D), and 100 nm in (E).

Proliferating LeptoDiakinesis germ cells zygo Pachytene Diplotene (oocytes) Sperm

A XX

No MSUC H3K9me2 H3K27me3 MES silencing H3K4me2 X activation

X chromosome

H3K9me2 H3K27me3 H3K4me2

Autosomes Proliferating LeptoMeiosis germ cells zygo Pachytene Diplotene I and II

Sperm

XO H3K9me2 MSUC H3K27me3 MES silencing H3K4me2 No X activation

X chromosome

H3K9me2 H3K27me3 H3K4me2

Autosomes B

DNA

H3K4me2 Pachytene nuclei

DNA H3K9me2 Pachytene nuclei

Eleanor M. Maine, Figure 2.2 The dynamic pattern of chromatin regulation in the germ line. (A) Schematic diagram of XX (upper) and XO (lower) germ cells as they enter and progress through prophase of meiosis I (see Fig. 2.1). Shaded bars indicate the relative level of three histone marks, H3K9me2, H3K27me3, and H3K4me2, on X chromosomes and autosomes. (B) Photomicrographs show H3K4me2 distribution in XX mid-pachytene nuclei and H3K9me2 distribution in XO mid-pachytene nuclei. In each image, DNA is labeled in red and the chromatin mark is labeled in green. Arrows indicate the X chromosomes. H3K4me2, a mark associated with actively expressed chromatin, is concentrated on the autosomes and not visible on the X chromosomes. Note that XO pachytene nuclei would have a similar H3K4me2 distribution at this time, which is prior to the late-pachytene X-linked gene activation observed in XX germ lines. H3K9me2, a mark associated with unpaired chromatin, is concentrated on the male X chromosome and barely detectable on the autosomes and hermaphrodite X chromosomes. H3K27me3, a mark associated with silent chromatin, is observed on X chromosomes and autosomes, but is particularly concentrated on Xs (adapted with permission from Strome and Kelly (2007). Copyright Cold Spring Harbor Laboratory Press).

A

B

High

MI

S

RV

Low C

D

N N

mf

Mitchell A. Watsky et al., Figure 4.3 Type I collagen gene expression and the colocalization of myofibroblasts and collagen in the infarcted myocardium. Normal heart expresses a low level of type I collagen mRNA (panel A). At day 7 post-MI, type I collagen mRNA is largely increased at the site of MI (panel B). Immunohistochemical aSMA detection reveals accumulated myofibroblasts (mf) at the infarcted myocardium at day 7 post-MI (panel C), which are colocalized with accumulated collagen (panel D, red). S, septum; RV, right ventricle; N, necrotic tissue. Panels C and D: 200. Reprinted with publisher’s permission from Sun et al. (2000).

A

B High

MI

nonMI Low

C

D

MI

E

F MF

MF

V1 V2

MF

Fb

Fb

MF

V1 V2

Mitchell A. Watsky et al., Figure 4.4 Renin, ACE, and Ang II receptor expression in the infarcted myocardium. By in situ hybridization, a high level of renin mRNA is expressed at the site of MI, but not in noninfarcted myocardium (nonMI) (panel A). Detected by autoradiography, high binding density of ACE (panel C) is observed at the site of MI. Immunohistochemistry reveals that myofibroblasts are responsible for the expression of renin (panel B, brown) and ACE (panel D, arrowheads). Panel E shows abundant myofibroblasts (brown) in the center of infarcted myocardium at week 4 post-MI. High Ang II receptor binding (black grains) is seen in myofibroblasts, whereas blood vessels (V1, V2) and fibroblasts (Fb) express low density of Ang II receptors. Panels A and B reprinted with publisher’s permission from Sun et al. (2001). Panels C and D reprinted with publisher’s permission from Sun et al. (1994). Panels E and F reprinted with publisher’s permission from Sun and Weber (1996c).