**Learning Materials in Biosciences**

Renato Paro Ueli Grossniklaus Raffaella Santoro Anton Wutz

# Introduction to Epigenetics

Learning Materials in Biosciences

Learning Materials in Biosciences textbooks compactly and concisely discuss a specifc biological, biomedical, biochemical, bioengineering or cell biologic topic. The textbooks in this series are based on lectures for upper-level undergraduates, master's and graduate students, presented and written by authoritative fgures in the feld at leading universities around the globe.

The titles are organized to guide the reader to a deeper understanding of the concepts covered.

Each textbook provides readers with fundamental insights into the subject and prepares them to independently pursue further thinking and research on the topic. Colored fgures, step-by-step protocols and take-home messages offer an accessible approach to learning and understanding.

In addition to being designed to beneft students, Learning Materials textbooks represent a valuable tool for lecturers and teachers, helping them to prepare their own respective coursework.

Renato Paro • Ueli Grossniklaus Rafaella Santoro • Anton Wutz

# **Introduction to Epigenetics**

Renato Paro Department of Biosystems Science and Engineering ETH Zürich Basel, Switzerland

Raffaella Santoro Department of Molecular Mechanisms of Disease University of Zürich Zürich, Switzerland

Ueli Grossniklaus Department of Plant and Microbial Biology University of Zürich Zürich, Switzerland

Anton Wutz Institute of Molecular Health Sciences D-BIOL, ETH Zürich Zürich, Switzerland

This book is an open access publication. ISSN 2509-6125 ISSN 2509-6133 (electronic) Learning Materials in Biosciences ISBN 978-3-030-68669-7 ISBN 978-3-030-68670-3 (eBook) https://doi.org/10.1007/978-3-030-68670-3

© The Editor(s) (if applicable) and The Author(s) 2021

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#### **Preface**

The DNA in a eukaryotic cell is the primary vehicle for transmitting traits to the progeny. DNA is, however, intimately connected to a number of factors controlling the expression of the genetic information. This partnership, termed chromatin, not only has a strong infuence on how genes are expressed but can build additional layers of information, which can be inherited in parallel to the genetic information. Epigenetics studies the processes behind the inheritance of traits that cannot be attributed to changes in the DNA sequence.

The epigenetic landscape of a cell contributes to its fate and diversity by "remembering" developmental decisions and by maintaining a stable and faithful memory over many cell divisions. The composition and structure of chromatin can mark and silence a single gene or even an entire chromosome while its allele or sister chromatid in the same nucleus remains active. These chromatin marks can be erased if required and new marks reestablished to adapt the cell to changing developmental or environmental conditions. Epigenetics attempts to understand what these marks are; how they are established, if required reversed; and, above all, how they are inherited to the next cell generation. DNA in itself, by marking bases with a chemical signal, adding a methyl-moiety on cytosine, can generate additional information beyond the genetic context. Notably, this methyl-moiety can be removed again without changing the sequence content but altering epigenetic pathways. DNA is packaged into a regular array of nucleosomes. These are composed of histones and their variants, which can as well be chemically modifed in a very intricate pattern. The histone marks are the result of a complex cellular machinery generating local signatures on the DNA sequence they package, thereby establishing an epigenetically transmissible state. Also, in this case, the marks can be eliminated or replaced by others, emphasizing the reversibility and fexibility of epigenetic control. A third component, regulatory RNA, plays an ever-increasing yet less well-understood role in epigenetic control, providing an important link between genetic and epigenetic information.

The interplay between these three major players generates a much more complex layering of transmissible information than the sequence of the bases alone could provide. Unlike the sequence information of DNA, the reversibility and fexibility of particular epigenetic marks ensures that genetic information can be interpreted in a much more context-dependent manner. For example, the process of organ and tissue regeneration requires substantial reprogramming of epigenetic control by changing and adapting cellular identities for tasks of repair and homeostasis. Most chromatinmodifying enzymes use metabolites as co-factors, intimately connecting the control of epigenetic mechanisms to the changing physiological needs of a cell. Eventually, if not appropriately maintained, epigenetic signatures can lead to malfunctions ranging from cancer development to neuronal disorders. Epigenetics plays a complementary role to genetics, touching many aspects of medicine, biotechnology, and plant biology.

This book is intended as an accompanying reading to the lecture "Epigenetics" taught by the authors at the ETH Zurich, Switzerland. The frst chapters provide an introduction to the biology of chromatin with all the components and cellular processes that establish the toolbox of epigenetic mechanisms. In the following chapters, complex epigenetic phenomena are illustrated by explaining the structures and principles of the underlying molecular mechanisms. Towards the end of the book, two chapters examine environmental infuences on epigenetic control and epigenetic misregulation in human disease.

I am very grateful to the authors of this book for their effort and commitment to realize this teaching support for the students. All of us highly appreciate the strong support and patience of Amrei Strehl and Bibhuti Sharma at Springer Nature's editorial offce. We apologize to many of our colleagues for the limited referencing of their cited work caused by space restrains. We hope this book will stimulate students and readers and further their interest and curiosity for the fascinating biology of epigenetics. Indeed, many fundamental questions and applications in epigenetics remain to be discovered and their mechanisms resolved in the future. This perspective makes research in this discipline a continued enjoyable and rewarding endeavor.

**Renato Paro**

Zürich, Switzerland

#### **Acknowledgments**

We are grateful for the curiosity and interest of our students. This book would not have been the same without their questions that brought common misconceptions and new viewpoints to our attention. R.P. thanks Yanrui Jiang for comments on the manuscripts and the ETH Zurich for fnancial support. U.G. is indebted to Hanspeter Schöb for administrative help in obtaining republication permissions. Work from R.S. was supported by the Swiss National Science Foundation (31003A\_173056), Krebsliga Schweiz (KFS-4527-08-2018-R), and a grant of the European Research Council (ERC AdG 787074 – NucleolusChromatin). Work by A.W. was supported by ETH Zurich and the Swiss National Science Foundation (31003A\_175643). Work by U.G. in this feld was supported by the University of Zürich, and grants of the European Research Council (ERC AdG 250358 – MEDEA) and the Swiss National Science Foundation (31003A\_179553, 310030B\_160336).

#### **Contents**

#### **Supplementary Information**

# **Biology of Chromatin**

#### **Contents**

#### **References – 28**

#### **What You Will Learn in This Chapter**

This chapter provides an introduction to chromatin. We will examine the organization of the genome into a nucleosomal structure. DNA is wrapped around a globular complex of 8 core histone proteins, two of each histone H2A, H2B, H3, and H4. This nucleosomal arrangement is the context in which information can be established along the sequence of the DNA for regulating different aspects of the chromosome, including transcription, DNA replication and repair processes, recombination, kinetochore function, and telomere function. Posttranslational modifcations of histone proteins and modifcations of DNA bases underlie chromatin-based epigenetic regulation. Enzymes that catalyze histone modifcations are considered writers. Conceptually, erasers remove these modifcations, and readers are proteins binding these modifcations and can target specifc functions. On a larger scale, the 3-dimensional (3D) organization of chromatin in the nucleus also contributes to gene regulation. Whereas chromosomes are condensed during mitosis and segregated during cell division, they occupy discrete volumes called chromosome territories during interphase. Looping or folding of DNA can bring regulatory elements including enhancers close to gene promoters. Recent techniques facilitate understanding of 3D contacts at high resolution. Lastly, chromatin is dynamic and changes in histone occupancy, histone modifcations, and accessibility of DNA contribute to epigenetic regulation.

#### **1.1 Introduction: Epigenetic Regulation in the Context of the Genome**

#### **1.1.1 Background: Gene Expression and Chromatin**

All organisms inherit traits from their parents, which are encoded in the succession of four bases in nucleic acids. All eukaryotic organisms possess deoxyribonucleic acid (DNA)-based genomes, whereby DNA comprises of antiparallel strands wound in a right-handed double helix. Although the sequence of bases as well as the 3D structure of the DNA helix contribute to the expression of traits, it is thought that the DNA sequence facilitates trait generation by the regulated expression of genes. Gene expression is not the only function of the genome, but replication and faithful inheritance of the genome to descendant somatic cells and the next generation, and evolutionary modifcations of the genome are principal functions of the heritable material. Importantly, a genome is not suffcient for generating an organism; this requires a suitable reader that can be represented by a cell or, in the case of higher organisms, an oocyte or zygote that must be from the same species as the genome. The reader implements molecular processes in the cell's nucleus that lead to the production of biomolecules – ribonucleic acids and other biosynthetically active molecules – that replenish the cells and maintain organismal tissues. Through feedback by transcription factors that are encoded in the genome and typically bind to specifc DNA sequences, complex regulatory circuits are established. In addition, the DNA itself is organized in a chromatin fber that facilitates the imposition of information along the DNA sequence. Chromatin also supports the transduction of this epigenetic information into regulatory processes that, in turn, affect transcription, replication, repair, and, in specialized cases, even changes of the DNA sequence. Reciprocal feedback of transcription, combinatorial activity of regulators, genome size, and temporal dynamics contribute to the complexity of gene regulatory networks. On the one hand, this has led to the evolution of developmental programs for complex body plans and, on the other hand, often makes the understanding of individual processes diffcult. Therefore, exploration of mechanism of chromatin regulation relies on well suited model systems that disambiguate the function of the components involved. Genomic imprinting and X chromosome inactivation are phenomena where expressed and repressed copies of individual genes are present in the same nucleoplasm and facilitate the study of chromatin-based regulation of transcription factor activity. In addition, ingenious approaches have been designed to analyze chromatinbased heritable expression states controlled by *Polycomb* (PcG) and *Trithorax* group (TrxG) complexes. This chapter contains an introduction to the organization and function of chromatin and provides a basis for understanding its role in regulating the cellular function of DNA sequences.

#### **1.1.2 Discovery of the Nucleosomal Structure of the Genome**

Linear arrays of spherical particles of about 70 Ångström in diameter were initially observed by electron microscopy of chromatin released from animal cells (. Fig. 1.1) (Olins and Olins 1974). The regular spacing of the spherical units and the fact that similar arrangements were found in many eukaryotes suggested a basic form of organization of the genome. Consistent with a regular structure, experiments using limited digestion of chromatin with nucleases and gel electrophoresis analysis revealed DNA fragments at regular intervals at multiples of 150 base pairs (bp). This is consistent with protection of around 150 bp of DNA from nuclease digestion, whereby nuclease cleavage occurs on the stretch of DNA that lies between spherical particles. Based on these studies, it has become clear that an understanding of the genome needs to consider the molecular components of nucleosomal structure.

#### **1.2 The Structure of the Nucleosome**

A nucleosome represents a single repeat unit for organizing the majority of the DNA of a cell.1 A nucleosome consists of 8 histone proteins and 146 bp of DNA, which is wrapped around them in two left-handed turns2 (. Fig. 1.2a–c). Histone proteins form an octamer complex that comprises positively charged surfaces formed by basic amino acid side chains, which interact with the negatively charged phosphate groups of the DNA backbone. The octamer is assembled from two of each histone H3/H4 and histone H2A/H2B dimers (. Fig. 1.2d). The histone H3/H4 dimers occupy the core of the nucleosome and the H2A/H2B dimers are more loosely associated

1 An exception to nucleosomal organization of eukaryotic genomes is found in male gametes of animals, where histones are largely replaced with protamine facilitating a distinct and highly compacted genome confguration.

2 Although it is clear that the large majority of DNA in cells is arranged in left-handed turns around the histone octamer, it has been suggested that right-handed DNA looping can be accommodated by the nucleosomal structure and has experimentally been produced *in vitro*.

..      **Fig. 1.1** Electronmicrographs of eukaryotic chromatin. Rat thymus chromatin **a** positive and **b** negative staining, and **c** chicken erythrocyte chromatin. (From Olins and Olins (1974))

(. Fig. 1.3). Histone proteins form a globular domain with a characteristic alphahelical arrangement called the histone fold (Luger et al. 1997). Flexible N-terminal regions of histone proteins, the so-called histone tails, associate more loosely with the nucleosome and are accessible for posttranslational modifcations.

#### **1.2.1 Histone Variants**

Although the nucleosomal structure appears homogenous, variation in histone composition can introduce different functionalities. Different variants of histones can be incorporated, whereby variations of histone H3 and histone H2A are common, and histones H2B and H4 appear to be predominantly canonical.

..      **Fig. 1.2** Scheme representing biologically relevant aspects of the nucleosome structure. **a** DNA forms a right-handed double helix with two strands aligned in opposite (antiparallel) orientation. The helix forms a major and minor groove, whereby DNA bases are more accessible from the major groove. **b** Winding of the DNA in left-handed turns over the histone octamere. **c** At a distance of approximately 70 bp, the two DNA loops face the same surface of the nucleosome. Two hypothetical factor bindingsites are indicated by the boxed "A". As one turn of the DNA is completed every 10 bp, both "A" boxes can be facing the surface through the major grove, depending on the position of the nucleosome. Nucleosome positioning along the DNA thereby allows for changes in binding-site geometry that can have a regulatory function. **d** A model for assembly of nucleosomes from histone dimers. DNA assembled into a nucleosomal structure is depicted at the bottom

Histone variants are encoded by a different set of genes and show slight variations in the amino acid sequence. In mammals, histone H3.1 and H3.2 are deposited during DNA replication in S-phase, whereas histone H3.3 is involved in histone exchange at active transcription units and at pericentric and telomeric heterochromatin. Incorporation of histone H3.3 outside of S-phase can be explained as it is the only histone H3 gene whose expression is not restricted to S-phase. Histones are very basic proteins and are associated with chaperones, or loaders, in the cell when they are not in a chromatin context (see book 7 Chap. 2 of Paro). Different histone loaders are involved in deposition of histone H3 variants. During S-phase, chromatin assembly factor 1 (CAF-1) incorporates histones H3.1 and H3.2 into DNA, whereas *histone cell cycle regulator* (HIRA) has a role in incorporating H3.3 into transcribed

..      **Fig. 1.3** A model-based on X-ray crystallographic structure determination reveals details of a nucleosome. DNA is wrapped around a protein core of 8 histone proteins (histone H3/H4 dimers, green and blue; histone H2AH2B dimers, red and yellow). Globular domains of histone proteins show helical folds and long, unstructured N-terminal peptides are pasted into the model as protruding from the core. (From Luger et al. (1997))

genes. At pericentric3 and telomeric heterochromatin, ATRX/DAXX is required for histone H3.3 incorporation. At the centromere of mammalian cells, canonical histone H3 is replaced by CENP-A, which contains a number of amino acid differences and adopts a more compact structure. It is thought that CENP-A contributes to the mechanical rigidity of centromeric chromatin that forms the basis of the kinetochore, where spindle microtubules attach for chromosome segregation in mitosis. The specifc loader JHURP is involved in CENP-A incorporation into centromeres.

Among histone H2A genes in mammals, the incorporation of the H2A.z variant is restricted to promoters of active genes. This is somehow surprising as H2A.z appears to represent the ancestral form of H2A and is the mammalian H2A gene that is most similar to the H2A gene of *Saccharomyces cerevisiae*. Therefore, the canonical H2A, which is found in the majority of mammalian chromatin, has a slightly different amino acid sequence to the ancestral histone. Additional variants of histone H2A have been correlated with gene activity. Whereas H2A.B is incorporated into chromatin over the transcription unit of active genes, macroH2A accumulates in

3 Pericentic chromatin lies next to the centromere and is normally in a repressed heterochromatic confguration.

silent chromatin. MacroH2A is a vertebrate-specifc histone H2A variant that contains a large C-terminal extension, which is commonly referred to as a macro-domain. MacroH2A is enriched at the inactive X chromosome of female mammals, consistent with its association with transcriptionally repressed chromatin (see book 7 Chap. 4 of Wutz). Through variation of the composition of histone proteins, the function of nucleosomes can be changed, which affects the function of the 146 bp long DNA associated with the nucleosome.

#### **1.3 Histone Modifcations**

Changes in histone composition are not the only way by which information can be added to nucleosomes. Histone proteins are the subject to posttranslational modifcations. In particular, the unstructured N-termini of histones can be modifed in a variety of ways, whereby the chemical spectrum of modifcations and the combinatorial complexity is high. Phosphorylation of serine, acetylation and methylation of lysine, and methylation of arginine residues are the most prominent histone modifcations. Notably, multiple methyl-groups can be added to lysines and arginines, thereby increasing the complexity. Transfer of a ubiquitin to lysines in histone H2A and H2B has also been described. Improvements of analytical techniques have revealed an increasing number of histone modifcations and it is likely that this trend will continue in the future. Posttranslational modifcations of histones and their functions are incompletely understood at present. However, the development of specifc antisera to detect modifed histones has provided key insights.

#### **1.3.1 Nomenclature for Histone Modifcations**

The chemical diversity of modifcations and the large number of acceptor sites on histone proteins makes it diffcult to describe the modifcation state of nucleosomes. To facilitate the precise and comprehensive documentation of experimental results and further theoretical elaborations, the scientifc community has adopted guidelines for a systematic nomenclature. The class of the histone protein is prefxed to the single character code and position4 of the amino acid carrying the modifcation, followed by an abbreviation of the chemical nature of the modifcation (. Fig. 1.4). For example, the short form for histone H3 carrying di-methylation of lysine 27 is H3K27me2. Similarly, H2AK119ub identifes histone H2A carrying a monoubiquitin modifcation on lysine 119. If the precise histone subtype is known this can also be incorporated, for example H3.3K4ac stands for histone H3.3 that is acetylated on lysine at position 4. This versatile nomenclature can easily be extended for multiple modifcations: H3K9me3S10p specifes a doubly modifed histone H3 with tri-methylation on lysine 9 and phosphorylation on serine 10.

4 Numbering of amino acids starts at the biochemical N-terminus, which is different from the methionine encoded by the start ATG of the histone gene due to the activity of methionine aminopetidase.

#### Nomenclatur of Histone modications

..      **Fig. 1.4** A schematic of short-form nomenclature for posttranslational histone modifcations

#### **1.3.2 Combinatorial Modifcations at Pericentric Heterochromatin**

Histone modifcations can be detected by suitable antibodies either using biochemical methods or by staining techniques followed by microscopy. The latter has been used in combination with fuorescence-labeled secondary antibody detection to analyze different histone modifcation states within the nucleus. Microscopy techniques for dual- or multi-color labeling have further facilitated the observation of colocalization of specifc protein complexes with histone modifcations. These approaches have been important to characterize different types of chromatin in the nucleus. When combined with fuorescent probes that hybridize with specifc DNA sequences, an assessment of the genomic context can be made, albeit at a modest resolution of several thousand bp and, hence, tens or hundreds of nucleosomes.

The pericentric repeats in mouse cells are observed as DNA-dense clusters that are brightly stained with DNA binding dyes such as DAPI.5 Association of H3K9me3 with this DAPI-dense region suggested a correlation of a specifc histone methylation mark with pericentric heterochromatin. Binding of H3K9me3 by the chromo-domain of heterochromatin protein 1 (HP1) leads to HP1 accumulation at the pericentric

5 DAPI (4′,6-diamidino-2-phenylindole) is a fuorescent dye that binds to DNA, allowing the detection of the relative density of DNA in the nucleus by fuorescence microscopy.

heterochromatin. The assay of co-localization with pericentric heterochromatin has been exploited to identify additional components. The curious and instructive anecdote of the human autoimmune serum MCA1 provides a concise understanding. Initial interest in the MCA1 antiserum was raised when it seemed to recognize centromeres in mitosis and, thus, suggested specifcity for a potential component of the kinetochore. Since MCA1 did not identify any know kinetochore-associated proteins, mass spectrometry was performed, leading to identifcation of none but histone proteins. Finally, the mystery was resolved when the specifcity of MCA1 was investigated on a panel of doubly modifed histone H3 peptides (Hirota et al. 2005). It turned out that MCA1 specifcally recognizes H3K9me3S10p, which occurs exclusively at pericentric heterochromatin in mitosis, when serine 10 is phosphorylated by cell cycle kinases, including polo-like kinase (PLK). The physiological effect of serine 10 phosphorylation is to displace HP1 from pericentric heterochromatin in mitosis. This shows that the interaction between HP1 and histone H3 is specifc for the H3K9me3 state and is disrupted by doubly modifed H3K9me3S10p. This result is important for two reasons. Firstly, it clearly demonstrates that combinations of histone modifcations can act in cellular regulation. Secondly, in hindsight, it became clear that antisera for specifc histone modifcations are sensitive to modifcations on neighboring amino acids. This has implications for the interpretation of results obtained with such immunoreagents as a failure to detect a signal does not always correlate with the absence of the modifcation but can also be caused by interference with a neighboring modifcation. Although it is assumed that in the majority of cases interference from neighboring modifcations can be neglected, there is no systematic study to ascertain the validity of this assumption.

Research on pericentric heterochromatin has also led to the identifcation of histone methyltransferases. Suppressor of position variegation 3-9 homologues 1 and 2 (Suvar3-9h1 and Suvar3-9h2) associate with pericentric chromatin and catalyze H3K9me3 (. Fig. 1.5). H3K9me3 acts as a binding site for HP1 and mediates the recruitment of the additional histone methyltransferases Suvar4-20h1 and Suvar4- 20h2 which, in turn, catalyze H4K20me3 at pericentric regions (Schotta et al. 2004). This example shows that modifcations from histone H3 can be propagated onto histone H4 within the same chromatin context. The observation that multiple histones and modifcations contribute to pericentric chromatin suggests a mechanism for the stability of heterochromatin.

#### **1.3.3 Histone Modifcations at High Resolution**

Practically, it is of interest to investigate which modifcations are present on nucleosomes at a given gene promoter. Knowing of the activity of genes, this can further identify correlations with histone modifcations that are generally associated with active or repressed chromatin. For this analysis, two prerequisites need to be fulflled. Firstly, suitable and specifc reagents are required to detect a particular modifcation of a histone. This has been facilitated by the development of a large array of antibodies that specifcally recognize histone modifcations. Secondly, the methodology needs to establish a link between modifed histones and the DNA sequence, with which these are associated. Chromatin immunoprecipitation (ChIP) does exactly allow for such an analysis (7 Method Box 1.1). Chromatin enriched for a defned

#### Combinatorial interaction of histone modications

The HMTs Suvar3-9h1 and Suvar3-9h2 catalyse tri-methylation of histone H3 lysine 9 in pericentric heterochromatin

..      **Fig. 1.5** A schematic of histone marks at pericentric heterochromatin. The histone methyltransferases Suv3-9h1 and Suv3-9h2 establish H3K9me3. The tri-methylated lysine 9 serves as a binding signal for HP1 to histone H3. HP1 binding and recruitment of the histone-methyltransferases Suv4- 20h1 and Suv4-20h2 leads to establishment of H4K20me1 (Schotta et al. 2004)

histone modifcation is isolated through immunoprecipitation using antibodies that recognize specifc histone modifcations. The DNA can then be purifed and analyzed by a variety of methods including hybridization to microarrays (ChIP-array), adapter ligation and sequencing (ChIPseq), PCR with gene-specifc primers, or by hybridization on dot blots. The latter has originally been used to investigate highly repeated parts of the genome, such as the pericentric repeats, whilst the former can provide genome-wide maps of histone modifcations at nucleosomal resolution.

#### **Method Box 1.1: Chromatin Immunoprecipitation**

Histone modifcations, and transcription factor binding, can be analysed at high resolution by chromatin immunoprecipitation (ChIP). For ChIP analysis, the DNA is cross-linked to histone proteins using formaldehyde, when chromatin is in its native form within cells. Subsequently, cells are lysed and chromatin is sheared either by ultrasound treatment or partial digestion with nucleases. Chromatin that is associated with a specifc histone modifcation or protein is enriched by immunoprecipitation with a specifc antibody. After purifcation, the chromatin associated DNA can be analysed by dot blot, gene specifc PCR, and next generation sequencing (. Box Fig. 1.1).

..      **Box Fig. 1.1** Analysis of chromatin modifcations by ChIP. A schematic shows an overview of the ChIP analysis (left) and an example view from a genome browser of a histone modifcation map obtained by next generation sequencing (right)

#### **1.3.4 Chromatin Modifcations Associated with Transcription Units**

From analyses in multiple cell types, correlation of gene activity and histone modifcations along transcription units has been performed. Some general rules can be deduced. Gene promoters are frequently marked by H3K4me3 and carry acetylated lysines on histone H4 and H3. The gene body of active genes is often marked by H3K36me3. The occurrence of H3K4me3 at the promoter and H3K36me3 over the gene body is a reliable indicator for transcription and has been used to identify new genes (. Fig. 1.6) whose transcripts would have been diffcult to detect due to their low abundance (Guttman et al. 2009).

In contrast, genes that are characterized by H3K9me2 or H3K9me3 tend to be transcriptionally inactive. Similarly, H3K27me3 and H2AK119ub are associated with the activity of PcG complexes that are well-known repressors of developmentally regulated genes (see book 7 Chap. 3 of Paro). In progenitor cells and embryonic stem cells (see book 7 Chap. 7 of Paro), these marks can also co-occur with the active mark H3K4me3. This observation has led to the discovery of bivalent chromatin at promoters with apparently active H3K4me3 and repressive H3K27me3 modifcations. It appears that this confguration is resolved when cells differentiate and yields active H4K4me3 or repressed H3K27me3 confgurations in separate cell

Characteristic histone modications of active genes

..      **Fig. 1.6** Illustration of ChIPseq analysis of the transcription unit of a hypothetical active gene. A peak of H4K4me3 enrichment overlaps the gene promoter whereas the transcription unit shows increasing H3K36me3 towards the 3′ end of the gene. If data is obtained at very high resolution, a gap in the H4K4me3 peak can be observed at the position of the transcription start site (TSS). At TSS one nucleosome is displaced and the DNA is bound by general transcription factors

lineages. Therefore, PcG complexes might pre-mark certain developmentally regulated genes before an expression state is committed, which is consistent with the developmental plasticity of progenitor cells (see book 7 Chaps. 3 and 7 of Paro).

#### **1.3.5 A Concept of Writers, Readers, and Erasers of Histone Modifcations**

Chemical reactions that lead to posttranslational modifcations of proteins use cofactors from metabolic pathways. Acetyl-Co-enzyme A, S-adenosyl-methionine, and ATP are used for the acetylation, methylation, and phosphorylation of histones, respectively (see book 7 Chap. 9 of Santoro). The corresponding enzymatic activities are referred to as histone acetyltransferases (HATs), histone methylases (HMTs), and histone kinases. Analogously, histone ubiquitin-transferases catalyze monoubiquitinylation. Posttranslational modifcations of proteins are frequent and can have different functions. Not all modifcations do possess an apparent physiological function and might be observed, to varying extents, as likely bystander reactions that the cell could not prevent. In a number of cases, highly specifc modifcations have been selected during evolution as signals that can have profound effects on the function of chromatin. Several histone modifcations have been implicated in the transcription of genes. Other modifcations act to establish a heritable signal for repression. Highly specifc functions have been identifed for particular methylation states of lysines in the N-termini of histone H3, whereas acetylation can be less specifc. Acetylation of lysines of histones is part of the chromatin assembly process and is thought to remove the positive charge from lysines, thereby preventing strong interactions with negatively charged phosphate groups of the DNA. Acetylation of lysines of histone H3 and H4 is also associated with active promoters and could similarly loosen the association of histones with DNA to facilitate accessibility. These observations suggest a charge effect of acetylation and biophysical mechanisms involved in regulating local chromatin accessibility. However, this is not the entire story.

Bromodomain6-containing proteins can bind to acetylated lysines and attract other regulators. Proteins that specifcally bind modifed histones are considered readers of histone modifcations. This group of proteins is particularly important for recognizing specifc methylations states of lysines in the N-terminus of histone H3. HP1 protein can associate with H3K9me3 through a chromodomain. A single interaction provides only little binding energy given the small size of the tri-methylmodifcation. However, HP1 can bind through a cooperative binding mechanism that enhances the binding of several HP1 proteins to longer stretches of H3K9me3 modifed chromatin. This explains the enrichment of HP1 on the pericentric regions of mouse chromosomes, where H3K9me3 is abundant. Similar to HP1, the Polycomb (Pc) protein contains a chromodomain which is specifc for H3K27me3 (see book 7 Chap. 3 of Paro). The relevance of chromodomains in cells has been confrmed by replacing the chromodomain of Pc by the one from HP1, whereby the protein was

6 Bromo- and chromodomains are defned based on sequence homology in a number of chromatinassociated proteins. They recognize posttranslational modifcations and sequence variation of histones in the context of the nucleosome.

redirected to pericentric chromatin. Similar protein domains exist for reading phosphorylated serines on histones. Importantly, 14-3-3 proteins have high specifcity for the doubly modifed histone H3S10pK14ac. This specifcity plays a role in gene activation. A switch from an inactive H3K9me3 and HP1-bound state to an active H3K9me3S10pK14ac state has been described during the activation of the cell cycle inhibitor p21, which is an important tumor suppressor gene (see 7 Chap. 8 of Santoro). Histone H3 serine 10 phosphorylation by ERK kinase activity interferes with HP1 binding to H3K9me3 and simultaneously facilitates the association of 14-3-3 proteins if lysine 14 is acetylated. This mechanism demonstrates how cell signaling, in combination with combinatorial histone modifcations, can contribute to complex gene regulatory mechanisms.

A last aspect of histone modifcations is their stability. Depending on their chemical nature, histone modifcations possess different lifetimes. Whereas phosphorylation is readily reversible through phosphatases, tri-methylated lysine modifcations can persist for extended periods. Histone deacetylases (HDACs) and histone demethylases (HDMs, or KDMs for lysine demethylases) remove acetyl- and methyl-groups from lysines, respectively. In addition, deubiquitinating enzymes contribute to the turnover of ubiquitin moieties. To date, mechanisms for the enzymatic removal of all histone modifcations have been described, suggesting that posttranslational modifcations of chromatin are dynamic and actively regulated by the cell. Proteins or complexes that remove histone modifcations are considered erasers of epigenetic information. Whereas some histone modifcations have been selected by evolution for a regulatory function other may be less important. Examples for the function of histone modifcations will be discussed in their physiological context in the following chapters of this book.

#### **1.4 DNA Modifcations**

Modifcations of histones illustrate how information can be added to the genome without changing the sequence of the DNA. This is exactly how we defned "epigenetics" in the opening of the book. Enzymatic activities for establishing and removing modifcations also illustrate how this information can be dynamically regulated during development or in response to external stimuli. However, we also had one expectation that is less easily explained: How can the potentially complex patterns of histone modifcations be transmitted through cell division? Upon replication of the DNA, twice the number of nucleosomes will need to be assembled and this necessitates that half of the histones are freshly produced whereas the other half keeps the previously established modifcation patterns (see book 7 Chap. 2 of Paro). How can the information be reestablished on the new histones? Or is it not restored but lost? There are good indications that epigenetic modifcations are maintained but the mechanisms appear to be complex and are poorly understood. To understand the problem of epigenetic heritability, we turn to a much simpler system where maintenance is mediated by a concise mechanism.

..      **Fig. 1.7** Schematic of the catalytic mechanism of DNA methylation. DNMTs attach to the 6 position of the pyrimidine ring of the cytosine and enter a covalent intermediate. Methylation of the 5 position induces a shift of electrons and releases the enzyme

#### **1.4.1 DNA Cytosine Methylation**

The cytosine base of DNA in animal and plant genomes can be chemically modifed to 5-methyl-cytosine (5mC). This modifcation is observed in the majority of animals but is conspicuously absent from some popular laboratory model organisms including the nematode *Caenorhabditis elegans*, the fy *Drosophila melanogaster*, and the yeasts *S. cerevisiae* and *Schizosaccharomyces pombe*. Although 5mC is not ubiquitous, it is widely distributed among animals7 and plants. In particular, DNA methylation is essential for mammalian development and has been extensively studied for its role in silencing tumor suppressor genes in certain types of human cancer (see book 7 Chap. 8 by Santoro).

5mC is catalyzed by the activity of DNA methyltransferases (DNMTs) that use S-adenosyl-methionine (SAM) as a methyl-donor. The catalytic center of mammalian DNMTs resembles the one of DNA methylases that are components of bacterial restriction systems. The reaction involves an attack at the 6 position of the cytosine ring by the thiol group of a cysteine, leading to the formation of a covalent bond between the cytosine and the DNMT. The reaction mechanism of DNMTs thereby comprises an intermediate that links DNMTs temporarily to DNA (. Fig. 1.7). This intermediate is subsequently resolved by transfer of a methyl-group from SAM to the 5 position of the cytosine ring, abstraction of a proton, and release of the DNMT enzyme. After release, DNMT enzymes are available for another reaction cycle. Derivatization of the ring system of a DNA base requires that the base is accessible. From structural analysis of bacterial DNA methylases, it has been proposed that the cytosine is fipped out from the DNA double helix by rotation of the phospho-deoxyribose backbone. In this way, the base can be inserted into a deep pocket where SAM and catalytic residues are in close contact to the 5 and 6 positions of the cytosine ring.

A variety of methods has been developed to analyze DNA methylation at specifc genes and genome-wide (7 Method Box 1.2). 5mC in mammalian genomes is preferentially found in the context of CG dinucleotides, but not on cytosines in other sequence contexts like CC, CA, and CT. The reason for this becomes clear when replication of the DNA and the maintenance of methylation patterns are considered. CG dinucleotides represent a symmetric confguration on the antiparallel DNA strand as C pairs with G and DNA strands have opposite polarity in the double helix.

7 DNA methylation has been observed in honey bees and ants suggesting that it is present in insects. The DNA methylation system was likely lost during the evolution of *D. melanogaster*.

..      **Fig. 1.8** A model for maintaining CpG methylation patterns in mammalian genomes. After a single round of DNA replication, hemi-methylated DNA is generated. Restoration of DNA methylation on the newly synthesized DNA strand leads to a heritable pattern of DNA methylation. DNMT1 is an enzyme that is targeted to hemi-methylated substrates and has maintenance DNMT activity. The original 5mC is not lost but unmethylated C is present in newly synthesized DNA. Hence, if CpG methylation patterns are not restored after 2 rounds of DNA replication, fully unmethylated (newly synthesized) DNA is expected (bottom). This leads to a loss of epigenetic information by passive, replication-dependent demethylation

If DNA with symmetrically methylated CG dinucleotides on both strands is replicated, two copies result, each having a methylated and an unmethylated (the newly synthesized) strand. Maintenance DNMT enzymes are recruited to hemi-methylated DNA to restore a fully symmetric methylation pattern (. Fig. 1.8). This mechanism requires the recognition of hemi-methylated CG dinucleotides and the activation of methylation activity to restore the pattern on the newly synthesized strand for a faithful maintenance of the epigenetic marks. This conceptually simple mechanism can explain the heritability of this particular epigenetic information through cell division. We will see shortly that the molecular details are not quite as simple but the overall concept is clear and easy to comprehend.

#### **Method Box 1.2: Analysis of DNA Modifcations**

DNA methylation can be detected by different methods. Bacterial restriction endonucleases that are inhibited by cytosine methylation can be used to digest genomic DNA. Electrophoretic size separation and hybridization with complementary probes (Southern blot) reveals unmethylated DNA at a lower molecular weight than the methylated DNA, which remains undigested. PCR with gene-specifc primers can

..      **Box Fig. 1.2** Analysis of 5mC by bisulfte conversion. **a** The reaction mechanism for the conversion of cytosine to uracil in the presence of bisulfte. **b** The conversion of 5mC to thymine is blocked under suitably chosen reaction conditions

also be used to detect methylated DNA. When the endonuclease cuts the unmethylated template, no PCR product is obtained. Suitable controls are required as interpretation relies on the completeness of the enzymatic reaction. By using restriction enzymes that recognize the same sequence but are not inhibited by cytosine methylation, the digestibility of the DNA can be confrmed. *Hpa*II and *Msp*I recognize CCGG, but only *Hpa*II is inhibited by C5mCGG.

5mC can also be detected by antibodies. Similar to ChIP, methylated DNA is precipitated using 5mC-specifc antibodies and can then be studied by a range of techniques including dot blots, PCR, and sequencing. Similar antisera have been developed for 5hmC. These antisera have further been used for immunofuorescence staining to observe the distribution of DNA modifcations in tissues and cells by microscopy techniques.

Bisulfte conversion allows to examine DNA methylation at single base resolution. Heating DNA in the presence of bisulfte leads to deamination of cytosine to uracil (. Box Fig. 1.2). Under suitable reaction conditions, the conversion of 5mC is not observed and, thus, cytosine remains unchanged. Sequencing of the converted DNA shows unmethylated cytosines as thymines, so that methylation can be identifed by comparison to a reference sequence.

Recently, nanopore sequencing and mass spectrometry have also been applied to detect and quantify DNA modifcations.

What would happen if methylation does not take place, possibly because the DNMT enzyme is inactive or unavailable? Well, after another round of cell division, genomes would emerge that have completely lost DNA methylation from both strands and, hence, the corresponding epigenetic information (. Fig. 1.8). We would expect this to happen to half of all cells that have descended from the last ancestor with fully symmetric methylation. Further divisions would lead to increased loss as methylation patterns would not be restored and the only remaining hemi-methylated DNA strands are the ones inherited from the last ancestral cell that was fully methylated.

In mammalian genomes, CG nucleotides are observed in less than the expected frequency.8 As a consequence, long stretches of DNA contain 5mC at a relatively low density. To explain this observation, evolutionary erosion of methylated CG dinucleotides has been suggested. Deamination of cytosine leads to uracil that is recognized as an illicit base in DNA and is effciently replaced. The deamination product of 5mC is thymine, which is a valid base in DNA and might not be repaired and, thus, can become fxed. If C to T mutations occur in the germline they can accumulate over evolutionary time and could explain the overall defciency of CG dinucleotides in the genome of species with DNA methylation. This view is further supported by the observation that the unmethylated genome of *D. melanogaster* does not show a defciency in CG dinucleotides.

Also in mammalian genomes, there are regions with the expected number of CG dinucleotides. These regions appear as islands of about 500–1000 bp of locally elevated CG density compared to the majority of the genome. These CpG islands9 have been associated with gene promoters and are normally devoid of any 5mC. However, methylation of cytosines in CpG islands does occur in specifc physiological contexts. CpG islands are methylated on the inactive X chromosome in female mammalian cells as a consequence of transcriptional repression (see book 7 Chap. 4 of Wutz). In addition, methylation of CpG island promoters of tumor suppressor genes is important in human tumors (see book 7 Chap. 8 of Santoro). It has been shown that loss of DNA methylation from the promoters of tumor suppressor genes can lead to their reactivation and cause cell cycle arrest and death of tumor cells. Therefore, DNMTs have been pursued as potential targets for treating human tumors (see book 7 Chap. 8 by Santoro).

The physiological function of DNA methylation has been explored by reverse genetic analysis in mice. Mice possess three genes with catalytic DNMT activity. DNMT1 is considered the maintenance DNMT and catalyzes most of the replicationcoupled cytosine methylation. This is the enzyme that we invoked in the simple mechanism for inheriting DNA methylation patterns earlier in this chapter. In addition, there are two enzymes, DNMT3A and DNMT3B, that are considered *de novo* DNMTs. These DNMTs are thought to newly establish DNA methylation patterns and are targeted by other factors to chromatin. All DNMTs possess characteristic sequence homology in their catalytic domains. In mice, DNMT1 and DNMT3B are essential for embryo development. Mutations of DNMT3B are also associated with the immunodefciency, chromosomal instability, and facial abnormalities (ICF) syndrome in humans, suggesting a contribution of DNMT3 to centromere function and

8 In a stretch of mammalian DNA, fewer CGs occur than any of the other possible dinucleotide combinations GC, AT, TA, AC, CA, AG, GA, CT, TC, GT, TG, AA, CC, GG, TT. In plants, CNG appears the sequence motif instead of CG. Like CG, CNG is symmetric on both DNA strands.

9 The term CpG islands, the "p" representing the phosphor-ribose linkage, is often used instead of CG islands to indicate that CpG dinucleotides and not CG base pairs are meant.

genomic stability. Part of the defects caused by loss of DNMT1 are derepression of retrovirus-like genomic elements, so called intra-cisternal A-particles (IAPs). Loss of DNMT1 also causes cell death of differentiated cells but might be tolerated in transformed cells to some extent. Surprisingly, early embryonic cells appear to tolerate a loss of DNA methylation. Studies have shown that DNA methylation is critical for gene regulation. In particular, gene regulation by genomic imprinting is intimately linked to cytosine methylation (see book 7 Chap. 5 of Grossniklaus).

Analyses of the effects of mutations on DNA methylation led to the identifcation of factors that are involved in the mammalian DNA methylation system. These include structural maintenance of chromosomes hinge domain 1 (SmcHD1), which contributes to DNA methylation and gene repression at promoters on the inactive X chromosome, the non-catalytic DNMT3L that acts together with DNMT3A in establishing DNA methylation patterns in the germline, and UHRF1 (also called NP95). UHRF1 is required for association of DNMT1 with the replication machinery. UHRF1 contains a domain with specifcity for hemi-methylated DNA. Further studies have led to the discovery that UHRF1 possesses catalytic activity and mediates ubiquitinylation of histone H3 lysines 23 and 18. H3K23ub and/or H3K18ub recruit DNMT1 to DNA where it changes the hemi-methylated to a fully methylated pattern. It is interesting to note that histone modifcations are an integral part of the maintenance pathway of DNA methylation patterns. We will see more overlap and crosstalk between DNA and histone modifcations in the chapters throughout this book.

The methyl-group of 5mC is located in the major groove of the DNA double helix where it can be accessed by readers of DNA methylation. Protein domains have been identifed that mediate specifc binding to methylated or unmethylated DNA. UHRF1 contains such a domain for recognizing hemi-methylated DNA. A family of DNA-binding proteins contains a methyl-DNA binding (MDB) domain. In this family of proteins, methyl-cytosine binding protein 2 (MeCP2) specifcally recognizes 5mC. The function of MeCP2 is not entirely clear but it appears to affect gene expression in a subtle way. Mutations affecting MeCP2 have been shown to cause RETT-syndrome in humans (. Table 1.1). RETT syndrome is named after its discoverer and characterizes a neurodevelopmental disorder. It mainly affects girls who initially appear to develop normally but, at about 1 year of age, regress and display neurologic symptoms that overlap with symptoms of autism. The mutation is in a heterozygous state and *MeCP2* is located on the X chromosome. Due to random X inactivation, only about half of the patients' cells lack *MeCP2* expression whereas the other half is phenotypically normal. It has been suggested that the presence of MeCP2-defcient cells might have a dominant effect disrupting brain functions, which is likely due to subtle changes in gene expression in neurons and glia cells. A mouse model has been established that recapitulates some neurological defects to study the disease mechanism. Experiments that allow the restoration of *MeCP2* function after symptoms have developed, suggest that neurologic phenotypes can be reversed (Guy et al. 2007). This fnding has spurred efforts to reactivate the intact copy of *MeCP2* in human patients where an intact copy of the gene resides on the inactive X chromosome. If these approaches were successful and *MeCP2* could be reactivated, possibly by removing DNA methylation, a treatment of this devastating disease could be found. However, caution is needed as the mouse model on which these hopes were based does not fully recapitulate all phenotypes that are observed in humans. Mice that lack MeCP2 completely are viable and show neurologic symptoms, whereas absence of MeCP2 in all cells is lethal in humans. Therefore, MeCP2 mutations are generally not observed in males.

A second way how DNA methylation can affect gene expression is by preventing the recognition of binding sites by transcription factors. One might think of a steric hindrance by methyl groups that stick out into the major grove of a DNA segment containing a binding motif. The transcription factor CCCTC-binding factor (CTCF) recognizes a motif containing cytosines that can be methylated. CTCF plays a role in chromatin organization and has been associated with the formation of chromatin boundaries and insulator function (see 7 Sect. 1.7.3 of this Chap., and book 7 Chap. 5 of Grossniklaus). CTCF binding is blocked by DNA methylation, which can have profound consequences for the genomic region.

Studies of DNA methylation in mouse development have revealed that early embryos lose most of their DNA methylation at the blastocyst stage (see book 7 Chap. 5 of Grossniklaus). Initially, the genomes of the gametes are characterized by substantial methylation, which is removed during the cleavage stages of preimplantation mouse development. Both active and passive (through DNA replication) demethylation mechanisms have been considered. It is interesting to note that the precise mechanism of enzymatic removal of 5mC from DNA has not yet been established. It is thought that DNA repair pathways play a role as it appears impossible to chemically remove the methyl-group from 5mC without opening the pyrimidine ring system. This eraser mechanism is therefore different from mechanisms of erasing histone modifcations and, thus, DNA methylation is considered a relatively stable epigenetic mark.

#### **1.4.2 DNA Cytosine Hydroxymethylation**

A fundamental advance in understanding DNA methylation in animal genomes has come from the discovery of a family of enzymes that convert 5mC in DNA. The ten eleven translocation (TET) family of proteins has three members in mice, TET1, TET2, and TET3. These are iron- and α-ketoglutarate-dependent dioxygenases that use molecular oxygen to oxidize the methyl-group of 5mC (. Fig. 1.9). The frst

..      **Fig. 1.9** Conversion of 5mC to 5hmC by TET dioxygenases. TET enzymes use oxygen to hydroxylate the methyl-group of 5mC. The reaction requires iron in oxidation state 2 [Fe(II)] and leads to the decarboxylation of α-ketoglutarate to succinate

product of this oxidation is 5-hydroxy-methyl-cytosine (5hmC). Further oxidation converts 5hmC to 5-formyl-cytosine (5fC), and 5-carboxy-cytosine (5caC). It has been suggested that recognition and removal of these oxidation products by the base excision DNA repair machinery could be a mechanism for active demethylation of DNA. Additional roles for different cytosine modifcations have been proposed in gene regulation, organization of nucleosomes, and the regulation of histone modifcations. It is important to recognize that the substrate for generating any of these modifcations is 5mC and, therefore, DNMTs are required. The dependence of some modifcations on a preceding reaction is interesting as a more complex regulation of epigenetic information might be established similar to an "AND" gate in Boolean logic. However, whether this aspect is used by cells is presently unclear. Also, constraints for the system of DNA modifcations come from the observation that DNA methylation is dispensable in some organisms (*D. melanogaster* and *C. elegans*), suggesting that evolutionary important processes are not strongly dependent on it.

TET1 was initially discovered at the breakpoint of chromosomal translocations associated with leukemia and gave the family its name: ten-eleven-translocation. Mutation of *Tet2* in mice leads to aberrant blood cell differentiation and leukemialike phenotypes (see book 7 Chap. 8 of Santoro). These observations suggest a role of TET dioxygenases in the regulation of gene expression during cell differentiation. The function of *Tet3* has been associated with the demethylation of the genome in mouse preimplantation embryos. TET3 is present in the oocyte as a maternally supplied protein. After fertilization TET3 converts 5mC on the paternal, i.e., spermderived, genome to 5hmC. In contrast, the maternal genome is protected and maintains 5mC in the zygote. This leads to a differential marking of maternal and paternal chromosomes by 5mC and 5hmC, respectively. Although, the differences are removed through the subsequent passive demethylation process during embryonic cleavage, it is important to note that parent-of-origin marking is initially genomewide. This is surprising considering that only a small number of imprinted genes maintain their parent-of-origin information throughout development.

#### **1.4.3 Interaction of DNA and Histone Modifcations**

At frst sight, the large number of chromatin modifcations might appear daunting to comprehend. Conceptually, it can be helpful to see different modifcations in the context of their biological functions. Thereby, the same chromatin modifcations might be established by different enzymes and contribute to different molecular pathways. Biochemical studies have identifed domains that recognize specifc chromatin modifcations and, thus, allow the prediction of successive modifcations if binding domains are part of enzymes. A number of rules can be considered to predict and describe the makeup and function of different states of chromatin.

CG-rich DNA appears to be recognized by the PcG complexes that establish H3K27me3 and H2AK119ub. Cytosine methylation of CpG islands prevents recruitment, suggesting an antagonism between DNA methylation and PcG proteins, at least in CG-rich DNA. H3K27me3 has been shown to coexist with H3K4me3 that marks promoters. Such H3K27me3 and H3K4me3 doubly marked chromatin is considered to carry a "bivalent" mark, indicating that it has an ambivalent state between active and inactive. Yet, active promoters are acetylated on H3K27 and H3K27ac and H3K27me3 cannot coexist on the very same lysine. This logic suggests a potential step for activation by blocking PcG activity through H3K27ac (see book 7 Chap. 3 of Paro). Notably, H3K9me3 does not appear to occur together with H3K4me3, such that a strong anti-correlation is observed. At pericentric heterochromatin, H3K9me3 is paired with H4K20me3 as we have seen earlier in this chapter. In addition, it is correlated with H3K4me1. Preventing methylation of histone H3 lysine 4 causes DNA *de novo* methylation. It has been shown that all mammalian DNMTs bind to H3K4 when it is unmodifed. Binding leads to structural changes that activate *de novo* DNMT activity. In addition, DNMT3A and DNMT3B bind to H3K36me2 and H3K36me3 that are associated with the gene body of active genes. It is thought that DNA methylation within the transcription unit helps to prevent spurious transcription initiation, thereby reducing transcriptional noise. Undoubtedly, future studies have to extend our understanding of functional pathways in chromatin but keeping a few general rules in mind is helpful to evaluate epigenomic data and to decipher its biological relevance.

#### **1.5 Chromatin Organization and Compartmentalization in the Cell Nucleus**

We have now taken a look at chromatin and identifed molecular characteristics that can carry epigenetic information along the DNA sequence. This has led to the view of a highly functional structure of chromatin. We end this chapter with a brief analysis of how chromatin is arranged in the cell nucleus. This aspect becomes important when one considers the large number of nucleosomes encompassing an animal genome. In the case of the diploid human genome, over 40 million nucleosomes will need to be assembled. This has implications of how genes can be identifed and how transcription factors can bind to their recognition sites. Transport is not the only issue. The RNA polymerase II complex that transcribes most messenger RNAs exerts force on the DNA fber. Transcribing in the order of 10,000 genes in a cell would lead to excessive movement within the nucleus. Observations from microscopy suggest

..      **Fig. 1.10** Organization of the cell nucleus. Super-resolution microscopy images of the cell nucleus have been annotated with prominent structures and compartments. (Adapted from Markaki et al. (2012))

that chromatin maintains a relatively stable distribution. Discrete foci are observed from pericentric heterochromatin, with similar chromatin appearing to cluster together. Also, chromosomes appear to occupy discrete volumes within the cell nucleus, referred to as chromosome territories (CTs). Although different CTs are in close contact, there is very little mixing of the majority of the DNA from different chromosomes. These observations constitute evidence that the chromatin fber is constrained and mechanisms exist that compartmentalize the nucleus.

Microscopy images of DNA-stained nuclei show the nucleoli as prominent structures (. Fig. 1.10). These are the locations where ribosomal RNA is transcribed by RNA polymerase I and the assembly of ribosomal subunits takes place. Nucleoli contain RNA and ribosomes at different stages of assembly. As they contain little DNA, they stain weakly with DNA dyes and appear dark under the microscope. Nucleoli are bordered by a rim of heterochromatin called perinucleolar heterochromatin. Throughout the nucleus, DNA staining is distributed with different brightness corresponding to nucleosome density. Notably, small holes appear throughout the DNA staining. These are channels within chromatin, which are thought to allow genes to be accessed by factors and transcripts to be transported (Markaki et al. 2012). Connections of these channels to nuclear pores have been observed in super-resolution images, illustrating how mRNAs might be exported from the interior of the nucleus. Channels and gaps within chromatin are referred to as interchromatin compartment (IC). Collectively, these observations suggest that processes might take place on the surface of chromatin with factor access and transport being dependent on the IC.

#### **1.5.1 Replication of Pericentric Heterochromatin Domains**

The use of 3D fuorescent *in situ* hybridization (FISH) with complementary DNA probes makes it possible to correlate nuclear sub-domains with repetitive sequences. Fluorescently labeled DNA probes can be used to detect minor and major satellite repeat DNA of the pericentric regions in fxed nuclei. These preparations can be further stained using antibodies for histone modifcations. From such experiments, it could be concluded that the pericentric repeats are organized into chromatin domains that are stained homogenously by H3K9me3 antisera. Thus, microscopy showed that pericentric heterochromatin is indeed marked with certain histone modifcations and associates with specifc proteins as we discussed earlier in this chapter. Microscopy can also be useful to gain insights into how heterochromatin behaves during DNA replication. Newly synthesized DNA can be marked by incorporation of modifed nucleotides such as bromo-deoxyuridine (BrdU), which can be visualized by specifc antisera. Replication of pericentric repeats appears to take place at the outer border of the heterochromatin domain. The replicated DNA then associates again with the domain of heterochromatin. As we have seen earlier, pericentric heterochromatin recruits enzymes that catalyze histone modifcations. It is, therefore, reasonable to predict that newly deposited histones on the replicated DNA will be targeted by histone modifying activities that are resident in pericentric heterochromatin. Restoration of the characteristic chromatin marks is accomplished during S-phase and chromosomes with faithfully marked pericentric repeats can then be segregated in mitosis. Therefore, self-association of pericentric heterochromatin can be regarded as a mechanistic basis for the heritability of histone modifcations. Although this is a plausible model, it is likely that the situation is more complex. Firstly, pericentric repeats are also subject to DNA methylation and, therefore, additional interactions might have to be taken into account to understand the mechanism. Secondly, it is presently not clear how self-association of pericentric chromatin is mediated and how it can be segregated from neighboring chromatin. Could it be an intrinsic property of chromatin modifcations? Are specifc H3K9me3 binding proteins mediating self-association by cooperative binding mechanisms? Potentially, additional factors could be involved in this process (see book 7 Chap. 3 of Paro). Anchoring of chromatin at non-chromatin structures has been proposed, including a nuclear matrix or nuclear scaffold. However, the components of these conceptually plausible structures remain to be revealed.

#### **1.5.2 Topologically Associating Domains**

Even in images from super-resolution microscopy, which can resolve details of 30–100 nm, a single voxel10 represents between 30 and 100 nucleosomes. Microscopy analysis is, therefore, on a different scale than our molecular understanding of reg-

10 A voxel represents the smallest volume that can be resolved in a 3D image. It is defned in analogy to a pixel, which represents a dot of a 2D picture.

ulatory elements. A typical 1 kb long promoter would be assembled into 7 nucleosomes. To bridge this gap, new methods have been developed. Chromatin conformation capture techniques (3C to HiC) aim to identify DNA fragments that are in close proximity of each other in the nucleus but might be far apart in the linear DNA sequence. The method preserves DNA contacts by cross-linking chromatin when it is in its native state in the cell (7 Method Box 1.3). Thereafter, chromatin is digested with restriction enzymes. Ligation of the ends of the cut DNA in very dilute conditions yields chimeric DNA fragments. DNA fragments that are frequently in close proximity in the nucleus have a higher probability to be crosslinked in the same chromatin particle and, hence, show an elevated number of chimeric fragments. Sequence determination of the ligation products and plotting the number of chimeric DNA fragments against their distance in the linear DNA sequence yields a contact frequency estimate. Using this approach, the *in vivo* 3D topology of chromosomes has been investigated. From these studies it has become apparent that chromosomes are made up of domains of interacting chromatin that are partitioned by boundaries between them. Intervals with high internal contact frequencies are termed topologically associating domains (TADs, 7 Method Box 1.3). Although TADs might share regulatory elements and be co-regulated, it is important to realize that 3C and related techniques usually investigate a large number – several thousands to millions – of cells and, therefore, only averages are observed. It is not always clear whether, in a given cell at a given time, all contacts take place. 3C techniques have been very successful in identifying gene regulatory elements such as enhancers that are located at a distance from their promoters but show a high contact frequency. Chromatin conformation data is, therefore, useful to determine regulatory interactions and possibly to identify genes that are coregulated by certain *cis*-regulatory elements. Recently, chromatin conformation capture-based techniques have been extended to single cell analysis. This has further contributed to our understanding of chromatin organization. From a study that used haploid G1 phase cells to make unambiguous assignments of DNA sequence contacts, it has become clear that a general subnuclear arrangement is adopted in all cells (Stevens et al. 2017). A-compartments predominantly consist of sequences that are correlated with active chromatin marks. These can be distinguished from B-compartments that contain largely inactive chromatin and frequently locate to the nuclear periphery. The precise winding and geometry of the chromatin fber is different in each cell. Therefore, chromatin in the nucleus is stochastically distributed at the smallest scale but becomes more deterministic as the scale increases. A potential mechanism for such a topology has been proposed as local self-organization of chromatin, whereby locally acting elements would affect chromatin structure in their proximity. The combined activity of many elements would then result

in the overall observed topology.

..      **Box Fig. 1.3** Chromatin conformation capture. **a** The 3D organization of chromatin is preserved by crosslinking protein complexes and DNA with formaldehyde. The DNA is then digested with nucleases into short fragments. Subsequent ligation of DNA ends from fragments that are crosslinked facilitates the capture of interactions between sequences that are distant in the linear sequence of the genome but have been in close proximity in the nucleus. Sequencing of the ligation products and analysis of the frequency of chimeric DNA can be used to calculate the frequency of interaction between distant genomic regions. **b** Organization of chromatin compartments in the nucleus (left) can be deduced from interaction frequencies. A matrix of color-coded interaction frequencies (middle) can visualize the interaction of sequences in the genome. The diagonal line that runs from top left to right bottom represents interactions of regions that are in close proximity in the DNA sequence and are, thus, closely linked by the DNA backbone. Topological associated domains (TADs) (right) are confned by borders that limit interactions. High frequencies of interactions are observed within TADs and little interactions are observed outside. Cohesin complexes have been associated with TAD borders. Schematic representation of A and B compartments is also shown

#### **1.5.3 Structural Maintenance of Chromosomes Complexes**

The association of enhancers with gene promoters and the organization of TADs raises the expectation for mechanisms that facilitate the winding and bending of the chromatin fber to achieve the observed topology. This aspect has been a long-standing question, which appeared diffcult to answer for many years. Recent evidence illustrates a surprising and very powerful mechanism involving protein complexes of the structural maintenance of chromosomes (SMC) family. SMC proteins associate with chromatin and have important roles in chromosome condensation and segregation during mitosis. Two distinct SMC complexes have been identifed, referred to as cohesin and condensin complexes in anticipation of their function in mediating sister chromatid cohesion and condensation of mitotic chromosomes, respectively. SMC proteins contain ATPase head domains that can hydrolyze ATP to exert motor function. Reconstituted recombinant condensin complexes (containing SMC1 and SMC3) have the ability to induce changes in the topology of fuorescently marked DNA molecules. This observation has been made in sophisticated microscopy-based biochemical assays (Ganji et al. 2018). DNA loops were formed when DNA fragments were incubated with condensin complexes and ATP. A similar function has subsequently been observed for a recombinant human cohesin complex (containing SMC2 and SMC4), when additional cohesin-associated factors were present (Kim et al. 2019). These experiments suggest the SMC family as important regulators of chromatin topology.

Cohesins have a role in the cell cycle where they mediate the cohesion of sister chromatids after replication of the chromosomal DNA until segregation at mitosis. In addition, cohesins have been found to be involved in gene regulation. Cohesins are loaded on chromatin in interphase and likely can slide along the chromatin fber, mediating loop formation. Some chromatin loops correlate with the presence of CTCF sites at the loop base (7 Method Box 1.3). CTCF binds sequences that are asymmetric and, therefore, have an orientation. CTCF sites at the base of chromatin loops are oriented towards each other, suggesting that CTCF might act in a directional manner to block cohesin sliding. A number of TAD boundaries can be correlated with CTCF binding sites. In addition, other molecules can also contribute to the localization of cohesins and possibly other SMC complexes. Therefore, the recent discovery of the DNA motor function of SMC complexes suggests a fundamental mechanism for establishing DNA topology at a larger scale that will certainly continue to attract attention in the future (Banigan and Mirny 2020).

#### **Take-Home Message**

#### **References**

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# **Chromatin Dynamics**

#### **Contents**

#### **What You Will Learn in This Chapter**

The nucleus of a eukaryotic cell is a very busy place. Not only during replication of the DNA, but at any time in the cell cycle specifc enzymes need access to genetic information to process reactions such as transcription and DNA repair. Yet, the nucleosomal structure of chromatin is primarily inhibitory to these processes and needs to be resolved in a highly orchestrated manner to allow developmental, organismal, and cell type-specifc nuclear activities. This chapter explains how nucleosomes organize and structure the genome by interacting with specifc DNA sequences. Variants of canonical histones can change the stability of the nucleosomal structure and also provide additional epigenetic layers of information. Chromatin remodeling complexes work locally to alter the regular beads-ona-string organization and provide access to transcription and other DNA processing factors. Conversely, factors like histone chaperones and highly precise templating and copying mechanisms are required for the reassembly of nucleosomes and reestablishment of the epigenetic landscape after passage of activities processing DNA sequence information. A very intricate molecular machinery ensures a highly dynamic yet heritable chromatin template.

#### **2.1 Basic Nuclear Activities**

The eukaryotic genome is packaged into a nucleosomal beads-on-a-string architecture (see book 7 Chap. 1 of Wutz). This allows the long but thin DNA fber to be protected, condensed, and organized in the cell nucleus. For example, tight packaging of the genome is used actively to suppress deleterious sequences to become mobilized, like transposable or retroviral elements. However, DNA acts as a template for a number of nuclear processes which require access to the genetic information and, thus, an open chromatin conformation. Some of the associated enzymes use, for example, the sequence of a DNA strand to accomplish their work. However, nucleosomal DNA is double-stranded requiring that the DNA fber be freed from histone packaging before these nuclear activities can be carried out. A number of mechanisms evolved to counter the primarily repressive nature of the nucleosome, permitting fexible and responsive nuclear activities.

A prime example in this respect is the process of *transcription*. The eukaryotic cell has three RNA polymerases, which are distinguished by the RNA products they produce. Their mode of action is similar, though, requiring the sequence information provided by an open DNA strand to synthesize the complementary RNA molecule (Cramer 2019). Before this process can start, gene activation mechanisms ensure that particular transcription factors (TFs) can access their specifc target sequences, further requiring a remodeling of the protective nucleosomal structure at their sites of action, like enhancers. Similarly, during the process of elongation, the RNA polymerase complex encounters a nucleosomal structure, which needs not only to be removed for the synthesis of the transcript but also reassembled in an epigenetically conform manner after passage. All these steps are in confict with the primarily static interaction of DNA and histones and require a dynamic chromatin structure responding to external signals, enzymatic processing, and mechanical forces.

The process of *DNA replication* uses the activity of DNA polymerases templating on single-stranded DNA the production of an exact copy of the genetic material (Hammond et al. 2017). As such, sites of origins, where replication starts, have to be freed of nucleosomes to allow the factors to bind DNA and assemble the replication machinery. Nucleosomes are removed ahead of the advancing replication fork and reassembled behind the site of replication. In addition, histone modifcations, DNA methylation, and other chromatin associated factors are reestablished to maintain the epigenetic landscape. Indeed, at the replication site, two distinct mechanisms of epigenetic inheritance are taking place, of which we do understand one very well. The beauty of the complementary DNA structure revealed immediately how this molecule can self-template its own copy. Conversely, we know much less about how heritable epigenetic information is duplicated and faithfully replaced after passage of the replication fork. While we understand comparatively well the duplication of DNA methylation patterns on newly synthesized DNA (see book 7 Chap. 1 of Wutz), we still lack a similarly comprehensive understanding of how protein-encoded information, including histone marks and histone variants is maintained in a heritable manner.

Other nuclear processes like *DNA repair* or *recombination* require the alignment of DNA sequences and, hence, a local removal of nucleosomal structures. Dedicated processes ensure fast repair of open damaged sites and the search factors required to align complementary genome sections for sequence exchange (Papamichos-Chronakis and Peterson 2013; Misteli and Soutoglou 2009).

Still other nuclear processes like the addition of *telomere sequences* to the end of chromosomes, the generation of anchoring points for the *segregation of chromosomes*, or the silencing of transposable or repeat elements in *heterochromatic sequences* are dependent on specifc chromatin structures for their functional performance. This shows that chromatin needs to be dynamically regulated to support nuclear processes during the cell cycle in a predictable and reliable manner. The dampening nature of nucleosomes has also a positive effect, however. Transcription factors have many more presumptive binding sites in the genome than are observed *in vivo*. In combination with highly processive enzymes like RNA polymerase II, this can result in spurious and unwanted genome transcription. Such "expression noise" is kept in check by the nucleosomal packaging and only regulated chromatin accessibility will allow the basic nuclear processes to trigger their activities at the specifc and required sites in the genome.

#### **2.2 Connecting Nucleosomes to DNA Sequence**

As packaging entities for an entire genome, it is clear that histones cannot rely on a similar DNA sequence-specifcity as transcription factors. Nevertheless, as a nucleosomal unit, histone proteins are intimately interacting with the DNA that bends around the histone core. This can only be achieved through distinctive chemical interactions between the nucleic acid and the histones. As such, nucleosome assembly is thermodynamically preferred for some sequences in comparison to others, suggesting that chromatin architecture is governed by the sequence of the genome (Parmar and Padinhateeri 2020; Hughes and Rando 2014; Struhl and Segal 2013). Certain sequence determinants, like particular dinucleotides, affect DNA bending and, in consequence, nucleosome formation. Optimal nucleosome formation occurs when bendable dinucleotides (AT and TA) occur on the face of the helical repeat (approximately every 10 bp) that directly interacts with histones (. Fig. 2.1).

..      **Fig. 2.1** Sequence determining nucleosomal position. **a** Nucleosome sequence preferences. Within the 147 bp that are wrapped around the histone octamer, there is a preference for distinctive dinucleotides that recur periodically at the DNA helical repeat and are known to facilitate the sharp bending of DNA around the nucleosome. These include ~10-bp periodic AA, TT, or TA dinucleotides that oscillate in phase with each other and out of phase with ~10-bp periodic GC dinucleotides. The linker regions exhibit a strong preference for sequences that resist DNA bending and, thus, disfavor nucleosome formation. Among these, poly(dA:dT) tracts and their variants are the most dominant and highly enriched in eukaryotic promoters. **b** Determinants of nucleosome positioning. (top) Nucleosome-depleted regions (NDRs) are generated either by poly(dA:dT) tracts and/or by transcription factors and the nucleosome remodeling complexes they recruit (see below). Gray circles indicate nucleosomes. (middle) Nucleosomes located at highly preferred positions (black circles) fanking the NDR are generated by nucleosome-remodeling complexes (for example, Isw2 and RSC, likely in a transcription-independent manner), and fne-tuned by the Pol II preinitiation complex (PIC) and associated factors. (bottom) Positioning of the more downstream nucleosomes depends on transcriptional elongation as well as the recruitment of nucleosome-remodeling activities (for example, Chd1 and Isw1) and histone chaperones by the elongating Pol II machinery. (From Struhl and Segal (2013))

The exact position of the histone octamer with respect to the 10-bp helical repeat of the DNA double helix is termed 'rotational positioning' and, thus, sequence is a critical determinant for the assembly of the nucleosome. However, protein-DNA interactions are not only recognizing base readout but are also dependent on the local shape structure of the DNA fber, which is determined by the local sequence of bases (Rohs et al. 2010). Local kinks in the DNA fber or variable width of the minor groove infuence the effciency of nucleosome build-up. Conversely, some homopolymeric sequences like poly(dA:dT) and poly(dG:dC) are rather stiff and are strongly inhibitory to nucleosome formation. Such sequences are often found at positions where nucleosome presence is reduced to allow transcription factors or polymerases access to enhancers or promoters, respectively. Hence, a combination of positively and negatively acting DNA sequences has a strong impact on the positioning of nucleosomes in the genome. Indeed, this information allowed predicting fairly accurately the position of nucleosomes in the yeast genome by computational methods. Similar attempts for more complex genomes have failed, probably because other factors might strongly infuence the assembly and positioning of nucleosomes (see below). This indicates that sequence-dependent effects might be less important for nucleosome positioning in higher organisms.

In addition to a local interdependence between DNA sequence and nucleosome position, a number of DNA regulatory elements with a strong impact on large-scale chromatin conformations have evolved. Highly repressed chromatin, like heterochromatin, is built by multiprotein complexes with a strong polymerization activity. They package the nucleosomal fber over an extended chromosomal region into a condensed and regular arrangement (see book 7 Chap. 1 of Wutz). Heterochromatic regions do not only show a local regular array of condensed nucleosomes but also a clustering in the 3D space of the nucleus. This highly repressive structure can potentially immerse nearby active genes. The propensity of heterochromatin to spread along the chromosome can be observed in position effect variegation (PEV). After chromosomal translocations, repressive heterochromatin can "spread" into regions with euchromatic genes inhibiting their normal regulated activity. Normally, the presence of regulatory elements, termed boundary elements or insulators, restrains this expansive behavior of heterochromatin (Valenzuela and Kamakaka 2006). In cases where these insulators are lost, a stochastic spreading of heterochromatin in neighboring euchromatic genes can be observed, resulting in a variable expression of these genes.

However, also at the local level, different chromatin domains and gene regulatory units need to be appropriately controlled such that they can exert their function not over the linear dimension of the DNA fber but in the true 3D space of the nucleus. At complex genetic loci, such insulator elements regulate the tissue-specifc and developmental-specifc interaction of distant enhancer elements with their corresponding promoter region. Additionally, such elements prevent the spreading of repressive chromatin, like PcG-chromatin (see 7 Chap. 3 of Paro), into neighboring active expression domains. Technically, this knowledge can be used by fanking a transgene with insulator elements generating a functionally independent domain protected from position effects at the insertion site of the transgene. Appropriate regulatory signaling can occur within the insulated-fanked domain as the insulators prevent accidental external chromatin interactions.

Factors like sequence-specifc CTCF bound to insulators do not only organize the local chromatin interactions, but, in conjunction with other chromatin-associated elements like cohesins, are also involved in large-scale chromatin loop formation or coordination of topologically associated domains (TADs; see book 7 Chap. 1 of Wutz)(Chen and Lei 2019; Pisignano et al. 2019). Hence, at many points in the genome the DNA sequence plays, through specifc DNA-protein interactions, an important structuring role in organizing chromatin and, consequently, contribute to the epigenetic landscape. Since every cell of a defned organism contains the same DNA, the question is how this apparent rigid structure of chromatin, nevertheless, permits a highly dynamic environment for the regulation of basic nuclear activities in a cell-type specifc manner. In the next section of this Chapter, we will discuss how nucleosome positioning is regulated.

#### **2.3 Nucleosome Remodeling**

#### **2.3.1 A Template for Transcription**

This nuclear process transcribes RNAs from genes that are located in different regions of the genomic templates. These transcription units range from ribosomal RNAs, tRNAs, and mRNAs to the newly discovered miRNAs and long non-coding RNAs that are involved in distinct biological processes. In all cases, the process of transcription is tightly controlled in a temporal and spatial manner. RNA polymerases have to pass through three phases: initiation, elongation, and termination (Cramer 2019). The initiation phase involves recognition of a promoter DNA sequence, opening of DNA strands, and synthesis of a short initial RNA oligomer (Cairns 2009). During the elongation phase, the polymerase uses the DNA template to synthesize the growing RNA chain in a processive manner. Finally, DNA and RNA are released during termination, and the polymerase can then be recycled and re-initiate transcription. This entire process is refected in a characteristic arrangement of nucleosomes and accompanying histone modifcations at the transcription unit. The promoter region is devoid of nucleosomes while the downstream region exerts regularly phased nucleosomes as a consequence of the repeated passage of the polymerase complex (. Fig. 2.1c, d).

Biochemically, the sequence of events can be ideally studied at an inducible gene, for example, a target gene activated by a nuclear hormone receptor (. Fig. 2.2). Under such conditions, the process can be started and analyzed in a synchronous manner by the addition of a hormone to a tissue culture. After hormone binding, the nuclear receptor dimerizes and enters the nucleus. With the help of accessory factors, it can bind to its DNA recognition site even in a nucleosomal context. To open chromatin structures, in a next step, remodeling factors are attracted (see book 7 Chap. 1 of Wutz and below). The unwound DNA can be bound by general TFs and, consequently, the transcriptional preinitiation complex can be loaded onto the promoter.

In many instances the cofactor SAGA is a multifunctional complex that facilitates transcription initiation in the context of chromatin. The TFIID complex also acts as a cofactor that is involved in promoter recognition. Both SAGA and TFIID can deliver the TATA box-binding protein (TBP) to promoters and stimulate assembly of the preinitiation complex. Eukaryotic RNA polymerases require a number of general transcription factors for initiation. Assembly of the polymerase on promoters to form a pre-initiation complex, for example, requires the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. In the case of Pol II, TFs do not target the polymerase directly but, rather, through interactions with cofactors, like the multiprotein complex Mediator. After DNA opening, the DNA template strand transitions into the active center of the polymerase. The template strand then directs *de novo* RNA chain initiation without the use of a priming oligonucleotide. Extension of the RNA to a critical length eventually triggers disassembly of the preinitiation complex, escape of the polymerase from the promoter, and formation of a stable elongation complex.

The Pol II elongation complex often pauses downstream of the transcription start site (Gaertner and Zeitlinger 2014; Adelman and Lis 2012). Paused Pol II can be stabilized by the elongation factors DSIF and NELF but may also terminate (. Fig. 2.3). Upon an additional positive signal, Pol II is released from pausing into active elongation by the kinase positive transcription elongation factor, (P-TEFb). P-TEFb phosphorylates Pol II, DRB sensitivity-inducing factor (DSIF), and negative elongation factor (NELF), and this leads to dissociation of NELF and recruitment of elongation factors that stimulate Pol II progression. A number of epigenetic processes control transcription directly at the site of paused Pol II. Silencing by the *Polycomb* group (PcG) proteins (see book 7 Chap. 3 of Paro) is the result of an inhibition of elongation by repressory PcG complexes. Transcriptional elongation is coupled to the processing of nascent RNA. This coupling requires the so-called

..      **Fig. 2.2** Key transition points during hormone signaling. After ligand binding, glucocorticoid receptor (GR) dimerizes, enters the nucleus, and binds target sequences such as the Glucocorticoid Response Element (GRE) within chromatin. The receptor binds co-regulators, such as NcoA1/SRC-1, NCoA2/TIF-2/GRP-1, and CBP/p300, which do not require remodeling to engage the receptor at the promoter. In the following step, these factors attract directly the BRG1 nucleosome remodeling complex SWI/SNF to the promoter leading to the formation of a nucleosome free region. This allows transcription factors such as NF1 and the octamer transcription factors (OTFs) as well as the TATA-binding protein (TBP) to engage with the promoter. Finally, mediator proteins are recruited and the preinitiation complex forms, leading to transcription by RNA Pol II. (From King et al. (2012))

C-terminal repeat domain (CTD). During elongation, the CTD is highly phosphorylated and recruits factors for post-transcriptional processing, like pre-mRNA capping, splicing, and also enzymes that modify histones. Indeed, the chromatin landscape at a gene appears to have a profound impact on post-transcriptional events and infuences for example the specifcity of splicing events. At the end of the transcribed region, Pol II transcribes the poly-adenylation site, which results in cleavage of the nascent RNA transcript and maturation of the mRNA.

At every point of the transcription cycle, the factors described above interact with a chromatin template and, therefore, require additional activities of partners to make DNA more accessible. Pioneer TFs can recognize their cognate DNA binding sequence in a nucleosomal context and open up chromatin by attracting histone acetylase complexes like SAGA that locally destabilize repressive nucleosomal structures. However, to make DNA accessible over an extended region, as required for example for the assembly of the large transcriptional pre-initiation complex, an energy dependent machinery in the form of ATP-dependent chromatin remodeling complexes is necessary.

#### **2.3.2 Chromatin Remodeling Complexes**

The process of remodeling involves reconfguring the histone-DNA interactions either by disrupting, reassembling, or moving nucleosomes (Sundaramoorthy 2019). Enzymes known as ATP-dependent chromatin remodeling complexes are capable of generating changes by binding to a single nucleosome and reconfguring it through utilizing the energy obtained from hydrolyzing ATP. Remodelers can (i) mediate nucleosome sliding in which the nucleosome position on DNA moves in either direction, (ii) generate access for TFs whereby histones remain bound but DNA becomes

..**Fig. 2.3** Establishment and release of paused RNA polymerase II. The promoter region is shown with the transcription start site (TSS). Factors that are involved in the establishment or release of paused Pol II, such as DRB sensitivity-inducing factor (DSIF; purple pentagon), negative elongation factor (NELF; orange oval), and positive transcription elongation factor b (P-TEFb; green diamond), are indicated. **a** Promoter opening often involves binding a sequence-specifc transcription factor (shown here as TF1) that brings in chromatin remodelers to remove nucleosomes from around the TSS and to render the promoter accessible for recruitment of the transcription machinery. **b** Pre-initiation complex formation involves the recruitment of a set of general transcription factors (GTFs) and Pol II, which is also facilitated by binding specifc transcription factors (also shown as TF1 for simplicity). This step precedes the initiation of RNA synthesis. **c** Pol II pausing occurs shortly after transcription initiation and involves the association of pausing factors DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). The paused Pol II is phosphorylated on its carboxy-terminal heptapeptide repeat domain (CTD). The region in which pausing takes place is indicated in the fgure. **d** Pause release is triggered by the recruitment of the positive transcription elongation factor b (P-TEFb kinase), either directly or indirectly by a transcription factor (shown here as TF2). P-TEFb kinase phosphorylates the DSIF–NELF complex to release paused Pol II and also targets the CTD. Phosphorylation of DSIF–NELF dissociates NELF from the elongation complex and transforms DSIF into a positive elongation factor that associates with Pol II throughout the gene. **e** In the presence of both TF1 and TF2, escape of the paused Pol II into productive elongation is rapidly followed by entry of another Pol II into the pause site, allowing for effcient RNA production. When the gene is activated, some nucleosome disruption is likely, as depicted by the lighter coloring of the downstream nucleosome. (From Adelman and Lis (2012))

..      **Fig. 2.4** The activities of chromatin remodeling complexes. Remodelers support assembly of a regular chromatin template by sliding deposited nucleosome and thereby providing space for new nucleosomes. Remodelers can make a binding site for a TF accessible by moving nucleosomes to the side (repositioning), by ejecting a complete nucleosome (eviction) or by partially freeing the DNA (unwrapping). In addition, remodelers can change the local composition of nucleosomes by ejecting a dimer or replacing histone dimers with corresponding variant forms (dimer exchange)

more accessible, (iii) induce eviction of the histone octamer, and (iv) replace a core histone with a variant histone (. Fig. 2.4) (Lorch and Kornberg 2017; Clapier and Cairns 2009). The general importance of these processes is also evident by the fact that components of chromatin remodeling complexes are found to be frequently mutated in cancers (see book 7 Chap. 8 of Santoro).

A shared property of this wide variety of effects rests on a common enzymatic function; an ATP-dependent DNA translocase activity that is utilized to break the histone-DNA contacts. This activity is carried out by an evolutionarily conserved catalytic core ATPase subunit. This remodeler ATPase subunit adopts a fold typical of the much larger superfamily 2 (SF2) DNA/RNA helicases and translocases. Analogous to SF2 family DNA translocases, the remodeler ATPase contains several highly conserved motifs, including WALKER A/B motifs, a DNA-binding cleft, and a site of ATP-binding that are all important for the DNA translocation activity. The ATPase of the remodeler differs from the SF2 DNA translocases in that they have much higher affnity for nucleosomes compared to naked DNA. An interesting aspect that emerged from the recently resolved CryoEM structures of various remodeler-nucleosome complexes is that remodelers unwrap outer turns of DNA from the nucleosome. This is a key step to minimize the number of histone-DNA contacts and maximize the output from ATP hydrolysis (. Fig. 2.5). Based on the shared domains/motifs within their ATPase subunit fne-tuning the remodeler

..      **Fig. 2.5** Model of INO80 nucleosome remodeling. The functional architecture of INO80 with motor (ATPase, red), grip (Arp complex, green) and grappler (orange) suggests that processive nucleosome sliding proceeds via a ratchet mechanism. Transient generation of loops between the motor and the grip could expose H2A–H2B for editing. Direct binding of H2A–H2B by the grappler sensorfoot could regulate variant- or modifcation-specifc editing. (From Eustermann et al. (2018))

ATPase function and defning its selectivity for nucleosomal substrates, the family of Snf2 ATPases is grouped into four different classes: SWI/SNF, ISWI, CHD/Mi-2, and INO80-SWR1.

*SWI/SNF remodelers* are regulators involved in a wide range of gene control processes in all cell types. Composed typically of 9–14 subunits, at least two forms of SWI/SNF-specifc Snf2 ATPases are present in most cell types with well-conserved signature domains: an acetyl lysine-binding bromo domain, a helicase-related SANT domain that binds actin-related protein (ARP), and the SF2 family helicase domain. In *Saccharomyces cerevisiae*, these two forms are termed as SWI-SNF (contains ATPase Swi2, also called Snf2) and RSC (contains ATPase Sth1). SWI/SNF complexes can oppose epigenetic silencing by PcG proteins (see book 7 Chap. 3 of Paro). In *Drosophila melanogaster*, PcG proteins were shown to maintain repression of *Hox* genes during embryogenesis, while the SWI/SNF complex promotes *Hox* gene activation. In mammalian cells, the SWI/SNF complexes are generally grouped into two types of complexes: hBAF complexes containing ATPase BRG1 or BRM and several alternate core subunits such as ARID1A or ARID1B and hPBAF (polybromo-associated BAF) complexes containing the BRG1 ATPase associated with factors such as PBRM1 and ARID2. Both are essential for cell survival and involved in a number of transcriptional processes.

The *ISWI (imitation switch) remodelers* were initially identifed in *Drosophila* and then subsequently observed in other species like yeast, *Xenopus*, and human. In *Drosophila*, the complex exists in at least four forms: ACF, NURF, CHRAC, and RSF. All four of these complexes possess the same ATPase protein ISWI but contain different associated subunits, which are likely important for specialized remodeler functions. In yeast *S. cerevisiae*, there are two distinct ISWI complexes, ISW1 and

ISW2. Mammals have two homologs of ISWI, SNF2L (encoded by the *SMARCA1* gene) and SNF2H (encoded by the *SMARCA5* gene). In mammals, fve ISWI complexes have been identifed: ACF, CHRAC, WICH, RSF, and NoRC. ISWI complexes are typically heterodimers and composed of an ISWI protein (SNF2H or SNF2L) and another subunit that provides specifc biological processes. The ISWI remodelers generate regularly spaced nucleosomal arrays, facilitating the deposition of histones onto DNA and participating in replication progression and transcriptional repression.

The members of the *chromodomain-helicase-DNA-binding domain (CHD)* family are characterized by the presence of tandem chromodomains in the N-terminal region of the ATPase and a C-terminal DNA-binding domain. This highly conserved family is divided into three subfamilies based on the presence of additional protein domains. The frst subfamily is defned by an additional DNA-binding domain located in the C-terminal region. This subfamily consists of the *S. cerevisiae* Chd1, the only CHD family member in yeast, as well as Chd1 and Chd2 in higher eukaryotes. The second subfamily includes the proteins Chd3 and Chd4 as well as *Drosophila* Mi-2, which are characterized by the presence of an additional N-terminal PHD domain. Members of the second subfamily are typically part of large multi-subunit complexes having besides chromatin remodeling functions, also histone deacetylation activities. The third subfamily contains the proteins Chd5– Chd9 and is characterized by additional functional motives in the C-terminal region, including paired BRK domains, a SANT-like domain, chromo domains, and a DNA-binding domain.

The remodelers of the *INO80 family* are the most recently identifed remodeling complexes. The multi-subunit INO80 (Inositol 80) and SWR1 (Sick with Rat8) are the subfamily members of the INO80 family. INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of +1 and −1 nucleosomes at promoter DNA. Recent cryo-EM studies resolved the interaction sites of INO80 with a nucleosome (see . Fig. 2.5) (Eustermann et al. 2018). Their analyses suggest a ratchet-like mechanism for how INO80 slides and possibly edits nucleosomes. INO80 unwraps and grips DNA and histones by multivalent interactions. The motor is positioned to pump DNA into the nucleosome against the Arp complex, which could hold onto DNA until a suffcient force is generated by multiple small steps of the motor. Such a tracking mechanism might create a DNA loop between the motor and the Arp complex. As a result, INO80 would move nucleosomes in larger steps of 10–20bp. The proposed ratchet mechanism explains DNA loop formation that results in large translocation steps, as well as the means for ATP-dependent H2A.Z → H2A exchange. The motor, stator and multivalent grip of INO80 enable highly processive sliding without release and large-scale reconfgurations such as editing while keeping the remainder of the nucleosome intact.

Central to each of these remodeler families is the ATPase domain that conducts DNA translocase activity through ATP hydrolysis. Accessory domains within the ATPase and auxiliary subunits in a multi-subunit remodeler fnetune the ATPase activity to the desired outcome of nucleosome remodeling. Hence, not only transcriptional processes require the moving capacity of the remodelers but all basic nuclear processes depend in one form or the other on this powerful competence to free DNA.

#### **Methods Box 2.1: Determining DNA Accessibility in a Chromatin Template**

As described in this chapter, the regulated access to the DNA template is a prerequisite for many nuclear enzymatic processes. In reverse, the appearance of accessible sites in chromatin could pinpoint to regions of regulation or active genomic processing. To this purpose, nuclei are treated with limited concentrations of nucleases like DNase I or micrococcal nuclease. Given the appropriate conditions, these enzymes will only cut exposed DNA but not DNA wrapped around histones or packaged in condensed or silenced nucleosomal structures like heterochromatin. Originally, open chromatin regions, termed DNase hypersensitive sites, could be determined at single genes. For example, after hormone induction, the chromatin rearrangements at the nuclear hormone binding sites and the accompanying changes at the promoter could be visualized by the appearance, over time, of DNase hypersensitive sites. Experimentally, after hormone induction of the cells in culture, the nuclei would be isolated and treated with nucleases under limiting conditions. The reaction would be stopped, the DNA isolated, its size further reduced with specifc restriction enzymes, and eventually separated on an agarose gel. Hybridization with probes encompassing the boundaries of the gene would reveal the sites at which the nuclease attack had reduced the original size of the restriction fragment. Comparison of hypersensitive sites from uninduced and induced cells could provide not only information about the potential location of regulatory sites and produce co-called "DNase footprints" but also give clues about how fast chromatin is opening to permit the associated nuclear process (. Box **Fig. 2.1**).

In the meantime, recognition of hypersensitive sites can be achieved on a genomewide scale. While the initial steps of limited nuclease attack to open chromatin conformations remains principally the same, the subsequent site recognition utilizes new high-throughput sequencing technologies. After nuclease digestion, fragments of different sizes appear; while nucleosome-packaged DNA, in general, produces larger fragments, regions with open chromatin or nucleosome depleted regions are cut more often, resulting in smaller fragments. Hence, a local high frequency of smaller fragments is an indication for a hypersensitive site in the genome.

A further improvement was achieved with ATAC-Seq (Assay for Transposase-Accessible Chromatin using Sequencing). Instead of DNase I, a mutated form of a transposase, Tn5, is used to fragment the chromatin. This enzyme not only cleaves DNA but also tags the resulting double-stranded DNA fragment with sequencing adaptors, which can be amplifed by PCR and subjected to high-throughput sequencing. Also in this case, the number of reads for a region correlates with how open that chromatin region is. The method has the advantage that much less starting material, compared to the other described methods, is required. Overall, the method needs less time as fewer steps are required and tricky enzyme titrations are not necessary.

These methods have provided substantial insight into how chromatin "breathes" under specifc physiological conditions, presenting a direct readout for chromatin dynamics. In all cases, however, the approach is dependent on nucleases which have an inherent albeit low DNA sequence specifcity. This could result in preferential site cleavage, which is not solely governed by the chromatin structure. These caveats have to be considered when generating and interpreting results measuring chromatin dynamics.

..      **Box Fig. 2.1** DNase I-hypersensitivity (DH) analysis at an inducible gene. The chromatin organization at the glucocorticoid-responsive enhancer element, 2.5 kb upstream of the promoter of the tyrosine aminotransferase gene, was probed in rat liver cells. Isolated nuclei of cells are digested with increasing amounts of DNase I. Digested genomic DNA is purifed, cleaved with a restriction enzyme, resolved by agarose gel electrophoresis, and subjected to Southern blotting. The DH sites are revealed by indirect end-labeling of restriction fragments through hybridization of a small radioactive probe (marked with arrows). In the uninduced state there are two DH sites at the promoter and one at 21 kb upstream. After hormone induction with corticosterone, nucleosomes are remodeled at the enhancer within 15 min. A new DH site appears 2.5 kb upstream of the transcriptional start site. This correlates with the binding of glucocorticoid receptors and a complex set of remodeling factors. On removal of the hormone ("washout"), the factors dissociate and canonical nucleosomes reform within 15 min and the 22.5-kb enhancer DH disappears. (From Becker and Workman (2013))

#### **2.4 Nucleosome Assembly**

The above section discussed processes evicting, moving, or replacing histones in nucleosomes and, thereby, liberating DNA for access to processing enzymes. However, the reverse process is as important in order to reconstitute the native nucleosomal template and the correct epigenetic landscape. This need is most evident at the time of DNA replication. The newly synthesized DNA has to be packaged and epigenetically marked in a regular and complementary manner. At the replication fork, a highly orchestrated mechanism not only templates and produces an identical copy of the DNA but also removes nucleosomes in front and reassembles histones into nucleosomes behind, now on two DNA fbers in an epigenetically impeccable state (Hammond et al. 2017).

#### **2.4.1 Histone Variants and Histone Chaperones**

Most histones are produced during the S-phase of the cell cycle. These DNA synthesis-coupled canonical histones provide the source for the repackaging of the two new chromatids. However, a number of DNA synthesis-independent histone variants are produced at any time in the cell cycle, with variable tasks in structuring the chromatin landscape (see also book 7 Chap. 1 of Wutz) (Talbert and Henikoff 2017). The incorporation of the centromeric H3 variant (known as CENPA in vertebrates or CENH3 in plants) instead of the canonical H3 into a nucleosome forms the foundational structure of centromeric chromatin. The replacement of human canonical H3.1 by the highly similar variant H3.3 has few apparent consequences. This variant has a primary role in repairing gaps in the chromatin landscape that result from nucleosome disruption occurring during basic nuclear processes. Several H2A variants affect gene expression: H2A.Z and H2A.B are implicated in transcription initiation whereas macroH2A in animals and H2A.W in plants seem to be associated with nucleosome immobility and transcriptional silencing. Hence, like DNA containing base variants like methyl-cytosine, also nucleosomes can alter their information surface by incorporating variants of the canonical histones. This provides an additional layer of fexibility and complexity for epigenetic processing as the variants can have an infuence on nucleosome stability and marking by chromatin modifers.

For the assembly of histones into nucleosomes *histone chaperones* are required (De Koning et al. 2007). Indeed, when histones are mixed with DNA in solution at physiological ionic strength, they precipitate unless chaperones help histones to fold properly, preventing their positive charges from engaging in nonspecifc interactions. The highly conserved histone chaperones are also necessary for escorting and depositing histones into chromatin and for histone storage. There are a number of different chaperones for cellular processes and pathways of histone assembly. Newly synthesized histones are bound by a series of chaperones in the cytosol before entering the nucleus. Once soluble histone complexes enter the nucleus, chromatin assembly factor 1 (CAF1) directs histone assembly behind the replication fork, whereas the histone regulator A (HIRA) complex directs assembly into chromatin independent of DNA synthesis (see below). In humans, CAF1 deposits canonical H3.1 and H3.2 during replication, whereas HIRA deposits the H3.3 variant throughout the cell cycle. Hence, any process in the cell requiring histone assembly utilizes the chaperoning capability of specifc complexes. . Figure 2.6 summarizes the identity and sites of action of these important factors in chromatin dynamics.

#### **2.4.2 The Replication Fork: Still the Major Enigma in Epigenetics**

The most outstanding cooperation between DNA, histones, histone chaperones, chromatin modifers, and the replication machinery is exemplifed at the replication fork (. Fig. 2.7) (Bellush and Whitehouse 2017; Hammond et al. 2017). The passage of genomic DNA through the small hole in the replicative helicase must result in the transient release of all DNA-binding proteins, including the disassembly of nucleosomes and, hence, the uncoupling of histone- and nucleosome-based epigenetic information from the DNA. Eventually, old nucleosomes, including many with histone variants and modifcations, are rapidly reassembled on the newly synthesized leading and lagging strands. Gaps between old nucleosomes are flled with nucleosomes comprising newly synthesized canonical histones. Histone chaperones support this dynamic process. Behind the replication fork deposition of new nucleosomes is mediated by the histone chaperone CAF1, which teams with the replication clamp proliferating cell nuclear antigen (PCNA) and travels with the replisome. CAF1 is a trimeric complex

..      **Fig. 2.6** Overview of histone deposition mechanisms. Newly synthesized histones are incorporated into chromatin via globally and locally acting mechanisms. A network of specialized histone chaperones controls histone delivery and deposition. The fgure provides an overview of replication-coupled and replication-independent pathways that require the incorporation of newly synthesized canonical histones and replacement variants, together with parental histone recycling. The histone chaperones that are implicated in each process are listed at the bottom. (From (Hammond et al. (2017))

..      **Fig. 2.7** Parental histone recycling during DNA replication. Evicted parental histones are randomly segregated to daughter DNA strands. It remains unknown whether other components of the DNA replication machinery have histone chaperone activity and whether deposition of old and new histones occurs by separate pathways (for details see text). (From Hammond et al. (2017))

responsible for assembling new canonical H3 nucleosomes behind the fork (see . Fig. 2.7). ASF1 interacts with H3-H4 by binding the homodimerization interface of H3 and can only present dimers, not tetramers, to CAF1. A single CAF1 molecule can bind two H3-H4 dimers or a crosslinked (H3-H4)2 tetramer, suggesting that CAF1 is capable of assembling and depositing (H3-H4)2 tetramers onto DNA. Hence, old nucleosomes might be randomly disassembled and reassembled from their dimer components behind the fork, resulting in histone octamers containing both new and old histones. However, new Histones H3 and H4 may have epigenetic marks added before chromatin assembly at the fork. It is thought that H3K9 monomethylation and H4K5 and H4K12 acetylation are commonly added moieties by modifying enzymes SetDBl and HAT1, respectively, in complex with histone chaperones (e.g., CAF1 or ASF1). Once chromatin has been assembled these acetylation marks might become removed by deacetylases and replaced by corresponding opposing marks like H3K9me3 in the case of heterochromatin. However, in animals, canonical H3.1-containing nucleosomes consist of nearly all old or all new (H3.1-H4)2 tetramers. This suggests that either old tetramers are transferred intact behind the fork or they are transiently disassembled and reassembled in a way that excludes the incorporation of new dimers. Transfer of tetramers or dimers occurs across the fork either to CAF1 via ASF1 or, perhaps with the assistance of FACT (Facilitates Chromatin Transcription), directly onto DNA. FACT may contribute to nucleosome disruption and histone recycling through interaction with the replicative helicase CMG complex (which includes CDC45-MCM2-7-GINS), polymerase α (Polα), and the single-strand binding replication protein A (RPA). MCM2 provides a binding platform for evicted H3-H4 tetramers and may facilitate their recycling directly, in collaboration with FACT, or as dimers with ASF1, which splits H3-H4 tetramers and forms a co-chaperone complex with MCM2.

The big question still is how, after the passage of the replication fork and the assembly of new and old nucleosomes, the local epigenetic landscape is reestablished (Singh 2014). A templating mechanism has to duplicate and appropriately pattern the new histones with the necessary modifcations. Given the vicinity to the parental histones, these could act as anchors and templates for readers, writers and erasers to appropriately tag the new histones (see book 7 Chap. 1 of Wutz). In light of the intricate patterns of modifcations observed at certain functional chromatin locations, this certainly begs the question which and how precisely a biochemical mechanism is capable to faithfully reproduce these instructions. A better understanding of this process represents certainly a major and ambitious challenge in epigenetic research.

#### **Take-Home Message**

#### **References**

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### **Cellular Memory**

#### **Contents**

#### **What You Will Learn in This Chapter**

The identity of cells in an organism is largely defned by their specifc transcriptional profle. During cell division, these profles need to be faithfully inherited to the daughter cells to ensure the maintenance of cell structure and function in a cell lineage. Here, you will learn how two groups of chromatin regulators, the *Polycomb* group (PcG) and the *Trithorax* group (TrxG), act in an antagonistic manner to maintain differential gene expression states. Members of the PcG cooperate in large multiprotein complexes to modify histones with repressive marks, resulting in condensed chromatin domains. Conversely, the TrxG proteins counteract the repressed domains by opening nucleosomal structures and establishing activating histone modifcations. PcG and TrxG proteins are evolutionary highly conserved and control diverse processes, such as the identity of stem cells in mammalian development to the process of vernalization in plants.

#### **3.1 Maintaining Cellular Fates**

Metazoan organisms reach their size and body complexity through a sequential series of cellular specifcations intermingled with phases of growth. During early embryonic stages, the body pattern is defned, the germ layers established, and the primordia for the organs delineated. Subsequently, during the process of differentiation the developmental fates are implemented to produce morphological features, organ structures, and functions fulflling the needs of survival and reproduction of the specifc organism. During all these development processes, cells also undergo a large number of divisions, with the requirement to retain the once established developmental fates1. To a large part, the fates and identities of cells are written in their differential patterns of gene expression. Hence, after each cell division, these unique transcriptional networks have to be reestablished to maintain cellular identities. This is not a trivial task given the extensive molecular and cellular rearrangements observed during the processes of genome replication and cellular division, where most regulatory proteins and chromatin factors are stripped from the DNA. However, complex mechanisms evolved to retain cellular identities in the wake of these processes, which ensure that developmental decisions are faithfully maintained. In other words, cells seem to have a *"cellular memory".*

How can the "memory" of cells be revealed? Transplantation experiments have demonstrated that cells and tissues react differently when moved to a foreign neighborhood, depending on their programmed state. Before being determined for a particular identity, transplanted cells often adapt to the identity of their new foreign neighbors. Conversely, when transplanted after having been determined, they retain their original identity even when exposed to a new developmental environment. This shows that there is a specifc period in the development of an organism in which cellular programs become fxed and irreversible (Wolpert et al. 2015).

1 Developmental fates are built through consecutive developmental restrictions. Among the frst are the allocations to the germ layers (ecdoderm, endoderm and mesoderm). Subsequently, cells receive information to be part of a specifc organ or tissue and fulfll a particular structural or functional role. Every step of these decisions have to be memorized and faithfully maintained.

#### **3.2 ·** PcG/TrxG System Maintaining Cellular Memory

In *Drosophila melanogaster*, at metamorphosis, larval cell are eliminated and adult precursor cells become differentiated during the subsequent pupal stage, giving rise to the organs and appendages of the adult fy. This process is triggered by the hormone 20-hydroxyecdysone (ecdysone). The burst of hormone induces a wellorchestrated cascade of transcriptional and post-transcriptional events leading to the differentiation of adult precursor (imaginal) cells. An example being the transformation of the morphologically inert imaginal disc cells which give rise to the highly specialized external cuticular structures, like wings, legs, or eyes. Concomitantly, larval cells and tissues which do not form part of the adult fy become marked for death and are histolyzed during metamorphosis. A classical experiment by Hadorn and collaborators demonstrated that imaginal discs maintain and remember their determined program (Hadorn 1968). Clusters of cells representing the primordia of imaginal discs are defned at early stages of embryogenesis. During subsequent embryonic and larval stages, theses clusters grow in size but do not differentiate. Only at metamorphosis, the cells implement their early defned transcriptional programs and differentiate into the structures and appendages that give rise to the external cuticle of the fy. Fragments of imaginal discs can be transplanted into the fuid of the abdomen of adult fies (hemolymph), which acts like an incubator, allowing the discs to grow in size but not to differentiate. This process can be repeated over many weeks and months, forcing the imaginal disc cells to undergo more cell divisions than they would have done during normal development of a fy. To observe whether the cells also retained their identity under these circumstances, they are transplanted back into a larva prior to metamorphosis. After undergoing metamorphosis, the hatched adult fy contains, in its hemolymph, differentiated cuticular tissue of the same identity as the original imaginal disc. This clearly demonstrates that cells can retain a memory of their programmed state even when placed in a foreign environment and undergoing many cell divisions.

Interestingly, Hadorn and collaborators observed rare cases where the transplanted imaginal disc fragments, after differentiation, produced cuticular structures other than those originally determined. Hence, cells can also "forget" and change fate. They termed this phenomenon *"transdetermination".* These changes of fate often mimicked phenotypes which had been observed in homeotic mutants. These are mutations in master regulatory genes required to establish the appropriate segmental identities in the fy (Lewis 1978). As such, it was argued that the maintenance of cellular memory is inherently connected to the faithful conservation of the specifc expression patterns of master developmental regulators.

#### **3.2 PcG/TrxG System Maintaining Cellular Memory**