《遗传学》课程教学资源（学科前沿）遗传与表观遗传 Epigenetic inheritance during the cell cycle

REVIEWS ocHR。 MATIN DYNAMICS Epigenetic inheritance during the cell cycle Aline V Probst*, Elaine dunleavy*and Genevieve Almouzni Abstract Studies that concern the mechanism of DNA replication have provided a major framework for understanding genetic transmission through multiple cell cycles. Recent work has begun to gain insight into possible means to ensure the stable transmission of information beyond just DNA, and has led to the concept of epigenetic inheritance. Considering chromatin-based information, key candidates have arisen as epigenetic marks, including DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA as well as higher-order chromatin organization. Understanding the dynamics and stability of these marks through the cell cycle is crucial in maintaining a given chromatin state The definition of epigenetics has received much atten- Recent research has highlighted DNA methylation tion, as attested by the number of recent publications-. as a bona fide epigenetic mark, and chromatin organiz 942to When originally coined by Waddington in 1942, the ation has emerged as a source of major candidates fo escribe how genes of a term epigenetics defined the causal mechanisms by carriers of information superimposed on that encoded genotype bring about a phenotype. Current definitions which the genes of a genotype bring about a phenotype. by DNA itself (BOX 1). In line with genetic information of epigenetics include the On revisiting this definition in 1987, Holliday applied epigenetic marks must be heritable to qualify as study of heritable changes in the term epigenetic to situations in which changes in true epigenetic information. Furthermore, in contrast to ne function that occur DNA methylation result in changes in gene activity. genetic information, which is meant to be highly stable, without aiteranons to the DnA Today, the most widely accepted definition -which epigenetic information reveals a certain level of plastic we adopt in this Review - designates epigenetics as ity and is inherently reversible. Therefore, one needs Centromere the study of heritable changes in genome function that to understand how a particular chromatin state that is A region of a chromosome that occur without alterations to the DNA sequence. This associated with a particular cell type can survive through is defined by the presence of a definition implies that particular states that define cell multiple cell divisions and, more specifically, how it can naot cresgeand histone hs identity are attained by heritable instructions -the face the dramatic perturbation that occurs during the functions as a platform for epigenetic marks that determine whether, when and passage of the replication fork in S phase. Depending kinetochore assembly during how particular genetic information will be read. The on the nature of the epigenetic mark, different strategies Itoss initial setting up of these epigenetic marks represents to restore or maintain epigenetic states operate, either an establishment phase. Here, we discuss epigenetic immediately following the disruptive event(that is, in a inheritance as the means to ensure the transmission replication-coupled manner)or in a manner that can be of epigenetic marks, once they are established, from separated in time from the disruptive event. mother to daughter cell and potentially from gener- The centromere is an attractive model to discuss the ation to generation. Therefore, epigenetic information concept of epigenetic inheritance during the cell cycle provides a form of memory that is necessary for the (BOX 2). It presents a paradigm for an epigeneticall maintenance of genome function, including both defined locus, because its functionality is not ensured the differential gene expression patter f a given cell by the underlying DNA sequence but rather by its lasticity. UMR21B Centre lineage(encompassing, for example, the maintenance of particular chromatin organization.Once established a cell identity after differentiation, position-effect varie- centromere organization and function have to be stably 6. rue d'Ulm,75231 Paris gation in Drosophila melanogaster, dosage compensa- maintained through multiple cell divisions to ensure tion and imprinting in mammals)and the propagation proper chromosome segregation. Given the essential These authors contributed of essential architectural features, such as telomeres and role of centromeres, the proper inheritance of epigenetic centromeres, that are required for cell viability or pro- marks, including the higher-order organization, which mail: almouzni@curie. fr liferation status. Any unscheduled compromise at these define centromeres, must endure chromatin disruption levels might lead to disease. during the passage of the replication fork or the repair 22009 Macmillan Publishers Limited All rights reserved

The definition of epigenetics has received much atten￾tion, as attested by the number of recent publications1–6. When originally coined by Waddington in 1942, the term epigenetics defined the causal mechanisms by which the genes of a genotype bring about a phenotype7 . On revisiting this definition in 1987, Holliday applied the term epigenetic to situations in which changes in DNA methylation result in changes in gene activity8 . Today, the most widely accepted definition — which we adopt in this Review — designates epigenetics as the study of heritable changes in genome function that occur without alterations to the DNA sequence1 . This definition implies that particular states that define cell identity are attained by heritable instructions — the epigenetic marks that determine whether, when and how particular genetic information will be read. The initial setting up of these epigenetic marks represents an establishment phase. Here, we discuss epigenetic inheritance as the means to ensure the transmission of epigenetic marks, once they are established, from mother to daughter cell and potentially from gener￾ation to generation. Therefore, epigenetic information provides a form of memory that is necessary for the maintenance of genome function, including both the differential gene expression patterns of a given cell lineage (encompassing, for example, the maintenance of a cell identity after differentiation, position­effect varie￾gation in Drosophila melanogaster, dosage compensa￾tion and imprinting in mammals) and the propagation of essential architectural features, such as telomeres and centromeres, that are required for cell viability or pro￾liferation status. Any unscheduled compromise at these levels might lead to disease. Recent research has highlighted DNA methylation as a bona fide epigenetic mark, and chromatin organiz￾ation has emerged as a source of major candidates for carriers of information superimposed on that encoded by DNA itself (BOX 1). In line with genetic information, epigenetic marks must be heritable to qualify as true epigenetic information. Furthermore, in contrast to genetic information, which is meant to be highly stable, epigenetic information reveals a certain level of plastic￾ity and is inherently reversible. Therefore, one needs to understand how a particular chromatin state that is associated with a particular cell type can survive through multiple cell divisions and, more specifically, how it can face the dramatic perturbation that occurs during the passage of the replication fork in S phase. Depending on the nature of the epigenetic mark, different strategies to restore or maintain epigenetic states operate, either immediately following the disruptive event (that is, in a replication­coupled manner) or in a manner that can be separated in time from the disruptive event. The centromere is an attractive model to discuss the concept of epigenetic inheritance during the cell cycle (BOX 2). It presents a paradigm for an epigenetically defined locus, because its functionality is not ensured by the underlying DNA sequence but rather by its particular chromatin organization9 . Once established, centromere organization and function have to be stably maintained through multiple cell divisions to ensure proper chromosome segregation. Given the essential role of centromeres, the proper inheritance of epigenetic marks, including the higher­order organization, which define centromeres, must endure chromatin disruption during the passage of the replication fork or the repair Laboratory of Nuclear Dynamics and Genome Plasticity, UMR218 Centre National de la Recherche Scientifique/Institut Curie, 26, rue d’Ulm, 75231 Paris Cedex 05, France. *These authors contributed equally to this work. Correspondence to G.A. e‑mail: almouzni@curie.fr doi:10.1038/nrm2640 Epigenetics This term was coined by Waddington in 1942 to describe how genes of a genotype bring about a phenotype. Current definitions of epigenetics include the study of heritable changes in gene function that occur without alterations to the DNA sequence. Centromere A region of a chromosome that is defined by the presence of a centromere-specific histone H3 variant (CenH3) and that functions as a platform for kinetochore assembly during mitosis. Epigenetic inheritance during the cell cycle Aline V. Probst*, Elaine Dunleavy* and Geneviève Almouzni Abstract | Studies that concern the mechanism of DNA replication have provided a major framework for understanding genetic transmission through multiple cell cycles. Recent work has begun to gain insight into possible means to ensure the stable transmission of information beyond just DNA, and has led to the concept of epigenetic inheritance. Considering chromatin-based information, key candidates have arisen as epigenetic marks, including DNA and histone modifications, histone variants, non-histone chromatin proteins, nuclear RNA as well as higher-order chromatin organization. Understanding the dynamics and stability of these marks through the cell cycle is crucial in maintaining a given chromatin state. Chromatin DynamiCs REVIEWS 192 | mARcH 2009 | VOlume 10 www.nature.com/reviews/molcellbio © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS Box 1 Candidate players for epigenetic inheritance transmission of information beyond the DNA sequence during cell division and from positon of epigenetic information is crucial for Nude maintaining differential gene expression chromatin patterns in differentiation, development and disease. Candidates for key players in different levels of chromatin include dna non-histone chromatin proteins that bind nuclear RNA and higher-order organization, as well as positional information. We need to distinguish between marks that reflect Nucleosome heat shock or damage)and those that are long-term instructions. These long-ter instructions might be inherited independently of the initial trigger, might qualify as epigenetic marks and could contribute to cellular memory2?. DNA wraps around a histone octamer that is 3 Histone modifications composed of one(H3-H4), tetramer capped by two H2A-H2B dimers. Together with the linker histone h1. this forms the nucleosome the basic building block of chromatin(see the figure). DNA itself is covalently modified by methylation of cytosine residues. Histones are also post-translationally modified (for example, by methylation(Me), acetylation (Ac)and phosphorylation (P)), and each mark constitutes a signal that is read alone or in combination with other modifications on the same or neighbouring histones as a histone code. Families of methyl-or histone-binding proteins decipher the regulatory information that is encoded by DNA methylation and histone marks. The presence of histone variants adds further complexity. Whereas the replicative variant H3.1 is DNA incorporated in a DNA synthesis-dependent manner, replacement variants, such as H3. 3 and the Histone variants specific histone H3 variant CenH3, are incorporated in a DNA synthesis-independent manner and result in nucleosomes with atypical stability. Nucleosomal chains fold into higher-order chromatin structures that are potentially organized with non-coding RNA components. The position of a particular chromosomal domain in the nucleus constitutes an additional level of instructions for gene expression. of damaged DNA. The basic rules that can be learnt variants that is either coupled or not coupled to DNA from the maintenance of a well-defined domain, such as replication. We discuss the maintenance of hetero the centromere, might further our understanding of the chromatin using the example of centromeres and show, general principles that underlie the inheritance of by means of reprogramming events that occur during epigenetic states. development, the reversibility of epigenetic marks and The actual nature and diversity of histone modifi- their dynamic Heterochromatin cations and modifiers, and histone variants, have A chromatin regon that been covered elsewhere, as have the challenges posed Inheritance at the replication fork remans oneens td an roughout to chromatin during replication and repair 21. Here, we In each cell cycle, the integrity of genetic and epigenetic characterized by a specific discuss the sophisticated mechanisms that have evolved information is challenged during DNA replication in order to facilitate the inheritance of epigenetic marks When DNA replicates, chromatin undergoes a wave of not only at the replication fork, but also at other stages disruption and subsequent restoration in the wake of the cell cycle. This Review provides an overview of of the passage of the replication fork. Whereas lineage tic state, resulting in an our current knowledge concerning the inheritance preservation requires the faithful maintenance of epi Tered cellular identity of DNA methylation, histone modifications and histone genetic marks, DNA replication also presents a window NATURE REVIEWS MOLECULAR CELL BIOLOGY 22009 Macmillan Publishers Limited All rights reserved

Nature Reviews | Molecular Cell Biology Nucleosome Structural RNA Higher-order chromatin Nuclear position Nucleus P DNA methylation DNA Histone modifications Histone variants Me Ac Ac Chromatin-binding protein Heterochromatin A chromatin region that remains condensed throughout the cell cycle and that is characterized by a specific chromatin signature. Reprogramming The induced reversal of an epigenetic state, resulting in an altered cellular identity. of damaged DNA. The basic rules that can be learnt from the maintenance of a well­defined domain, such as the centromere, might further our understanding of the general principles that underlie the inheritance of epigenetic states. The actual nature and diversity of histone modifi￾cations and modifiers10, and histone variants11, have been covered elsewhere, as have the challenges posed to chromatin during replication and repair12,13. Here, we discuss the sophisticated mechanisms that have evolved in order to facilitate the inheritance of epigenetic marks not only at the replication fork, but also at other stages of the cell cycle. This Review provides an overview of our current knowledge concerning the inheritance of DNA methylation, histone modifications and histone variants that is either coupled or not coupled to DNA replication. We discuss the maintenance of hetero￾chromatin using the example of centromeres and show, by means of reprogramming events that occur during development, the reversibility of epigenetic marks and their dynamics. inheritance at the replication fork In each cell cycle, the integrity of genetic and epigenetic information is challenged during DNA replication. When DNA replicates, chromatin undergoes a wave of disruption and subsequent restoration in the wake of the passage of the replication fork. Whereas lineage preservation requires the faithful maintenance of epi￾genetic marks, DNA replication also presents a window Box 1 | Candidate players for epigenetic inheritance Epigenetic inheritance refers to the transmission of information beyond the DNA sequence during cell division and from one generation to the next1,3. Inheritance of epigenetic information is crucial for maintaining differential gene expression patterns in differentiation, development and disease. Candidates for key players in epigenetic inheritance that are situated of different levels of chromatin include DNA and histone modifications, histone variants, non-histone chromatin proteins that bind directly to DNA or to histone modifications, nuclear RNA and higher-order organization, as well as positional information. We need to distinguish between marks that reflect short-term instructions and can quickly revert in response to a signal (for example, heat shock or damage) and those that are long-term instructions. These long-term instructions might be inherited independently of the initial trigger, might qualify as epigenetic marks and could contribute to cellular memory2 . DNA wraps around a histone octamer that is composed of one (H3–H4)2 tetramer capped by two H2A–H2B dimers. Together with the linker histone H1, this forms the nucleosome — the basic building block of chromatin (see the figure). DNA itself is covalently modified by methylation of cytosine residues. Histones are also post-translationally modified (for example, by methylation (Me), acetylation (Ac) and phosphorylation (P)), and each mark constitutes a signal that is read alone or in combination with other modifications on the same or neighbouring histones as a ‘histone code’. Families of methyl- or histone-binding proteins decipher the regulatory information that is encoded by DNA methylation and histone marks. The presence of histone variants adds further complexity. Whereas the replicative variant H3.1 is incorporated in a DNA synthesis-dependent manner, replacement variants, such as H3.3 and the centromere￾specific histone H3 variant CenH3, are incorporated in a DNA synthesis-independent manner and result in nucleosomes with atypical stability. Nucleosomal chains fold into higher-order chromatin structures that are potentially organized with non-coding RNA components. The position of a particular chromosomal domain in the nucleus constitutes an additional level of instructions for gene expression. REVIEWS NATuRe ReVIeWS | Molecular cell Biology VOlume 10 | mARcH 2009 | 193 © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS entromeres are key chromosomal elements that are responsible for correct chromosome segregation at each cell division". Whereas in budding yeast the incorporation of the centromere-specific histone H3 variant CenH3 is determined particular DNA sequence, such a sequence requirement has been lost during evolution. At most centromeres, rapidly evolving repetitive sequences are found and centromere function is determined by chromatin organization and the esence of CenH3. Therefore, centromeres are a paradigm for an epigenetically defined domain. They consist of a central main called the inner centromere or centric heterochromatin which is at the basis of kinetochore formation and where CenH3 is incorporated (see the figure, part a). The adjacent pericentric heterochromatin(pHC) contributes to centromere sister chromatid cohesion 03 0424 Pericentric heterochromatin cell cycle and individual pericentromeres come together into large clusters called chromocentre", as shown by DNA fluorescence in situ hybridization( FISH) for pericentric satellite repeats in mouse embryonic fibroblasts(see the figure, art b). At the molecular level, pericentric heterochromatin is characterized by extensive DNA methylation and specific istone methylation marks, such as dimethylated and trimethylated H3K9 (H3K9meZ and H3K9me3, respectively ), that e bound by heterochromatin protein 1(HP1; see the figure, part c). There are three HP1 proteins in mammals HPla, HP1p and HPly(also known as CBX5, CBX1 and CBX3, respectively). RNA interference(RNAi)contributes to heterochromatin integrity in fission yeast and plants however, a direct connection in flies and mammalian cells is so far king. Not every epigenetic mark is present at pericentric heterochromatin in all model organisms. Scale bar, 5 um (Heterochromatin(CenTs( Heterochromatin O DAPI c Centromere characteristics in different organisms Organis DNA sequence Centromere- H3K9 HPl RNAi specific H3 variant methylation methylation Cse4 Schizosaccharomyces pombe No Yes Yes Drosophila melanogaster Arabidopsis thaliana HTR12 Mammals CENP-A Yes of opportunity for changes in epigenetic states to occur antigen(PCNA), which is loaded onto both strands uring differentiation and development. Thus, refined Thus, PCNA provides an important link between the two mechanisms have evolved to ensure stability through the strands, and folding of the two strands in space might concerted transmission of genetic and epigenetic infor- further ensure the coupling of replication mechanisms mation at the replication fork, and to ensure plasticity on both leading and lagging strand(FIG.Ia).When that allows the desired switches during development. considering epigenetic marks, in addition to duplicating Understanding how to deal with this dual require- DNA, it is important to evaluate how DNA methylation, ment is a fascinating issue into which we have begun histone deposition and histone marks are connected to to gain insight. the replication machinery. In addition to its role in dNA synthesis, PCNA might also link DNA synthesis and the Inheritance of DNA methylation during replication. inheritance of epigenetic marks", as suggested by Since the first proposal that genetic information is the early observation that particular mutations in PCNA replicated in a semi-conservative manner 4, much has suppress position-effect variegation in D. melanogaster been learned about the enzymes and machinery at work Furthermore, PCNA interacts with many chromatin during replication s. However, it is only beginning to assembly and chromatin-modifying factors213, 19 212 emerge how, at the replication fork, the inheritance of (FIG. Ic; see below). In addition to PCNA, other factors genetic and epigenetic information can be coupled and are likely to contribute to the crosstalk between the inher how components of the DNA replication machinery itance of genetic and epigenetic information. Indeed, potentially crosstalk with all of the aspects of inheritance the minichromosome maintenance(MCM) complex, beyond the DNA sequence which is the putative replicative helicase, interacts with DNA replication proceeds in an asymmetric manner the histone chaperone anti-silencing function 1(ASFI; see Histone chaperone with continuous synthesis on the leading strand and below)2, which is proposed to coordinate histone flow A tactor that associates with discontinuous synthesis on the lagging strand (FIG. la, b). on parental and daughter strands histones and stimulates a This synthesis is catalysed by specialized DNA poly Similar to the semi-conservative inheritance ofdna merases on each strand. DNA polymerases are assisted sequences, patterns of symmetrical DNA methylation at by the DNA processivity factor proliferating cell nuclear CpG(cytosine followed by guanine)sites are transmitted 194 MARCH 2009 I VOLUME 10 22009 Macmillan Publishers Limited All rights reserved

Nature Reviews | Molecular Cell Biology Organism DNA sequence requirement Centromere￾specific H3 variant HP1 RNAi pathway DNA methylation H3K9 methylation Saccharomyces cerevisiae Yes Cse4 N No No o No Schizosaccharomyces pombe No Cnp1 No Yes Yes Yes Drosophila melanogaster No CID No Yes Yes Yes? Arabidopsis thaliana No HTR12 Yes Yes No Yes Mammals No CENP-A Y Yes Yes es Unknown c Centromere characteristics in different organisms b Mouse nuclei Heterochromatin Pericentric Centric Pericentric CenH3 Heterochromatin a DAPI pHC Histone chaperone A factor that associates with histones and stimulates a reaction that involves histone transfer without being part of the final product. of opportunity for changes in epigenetic states to occur during differentiation and development. Thus, refined mechanisms have evolved to ensure stability through the concerted transmission of genetic and epigenetic infor￾mation at the replication fork, and to ensure plasticity that allows the desired switches during development. understanding how to deal with this dual require￾ment is a fascinating issue into which we have begun to gain insight. Inheritance of DNA methylation during replication. Since the first proposal that genetic information is replicated in a semi­conservative manner14, much has been learned about the enzymes and machinery at work during replication15. However, it is only beginning to emerge how, at the replication fork, the inheritance of genetic and epigenetic information can be coupled and how components of the DNA replication machinery potentially crosstalk with all of the aspects of inheritance beyond the DNA sequence. DNA replication proceeds in an asymmetric manner with continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand (FIG. 1a,b). This synthesis is catalysed by specialized DNA poly￾merases on each strand16. DNA polymerases are assisted by the DNA processivity factor proliferating cell nuclear antigen (PcNA)17 , which is loaded onto both strands. Thus, PcNA provides an important link between the two strands, and folding of the two strands in space might further ensure the coupling of replication mechanisms on both leading and lagging strand18 (FIG. 1a). When considering epigenetic marks, in addition to duplicating DNA, it is important to evaluate how DNA methylation, histone deposition and histone marks are connected to the replication machinery. In addition to its role in DNA synthesis, PcNA might also link DNA synthesis and the inheritance of epigenetic marks19, as suggested by the early observation that particular mutations in PcNA suppress position­effect variegation in D. melanogaster20. Furthermore, PcNA interacts with many chromatin￾assembly and chromatin­modifying factors12,13,19,21,22 (FIG. 1c; see below). In addition to PcNA, other factors are likely to contribute to the crosstalk between the inher￾itance of genetic and epigenetic information. Indeed, the minichromosome maintenance (mcm) complex, which is the putative replicative helicase, interacts with the histone chaperone anti­silencing function 1 (ASF1; see below)23, which is proposed to coordinate histone flow on parental and daughter strands. Similar to the semi­conservative inheritance of DNA sequences, patterns of symmetrical DNA methylation at cpG (cytosine followed by guanine) sites are transmitted Box 2 | heterochromatin at centromeres Centromeres are key chromosomal elements that are responsible for correct chromosome segregation at each cell division94. Whereas in budding yeast the incorporation of the centromere-specific histone H3 variant CenH3 is determined by a particular DNA sequence, such a sequence requirement has been lost during evolution9 . At most centromeres, rapidly evolving repetitive sequences are found and centromere function is determined by chromatin organization and the presence of CenH3. Therefore, centromeres are a paradigm for an epigenetically defined domain. They consist of a central domain, called the inner centromere or centric heterochromatin, which is at the basis of kinetochore formation and where CenH3 is incorporated (see the figure, part a). The adjacent pericentric heterochromatin (pHC) contributes to centromere function by ensuring sister chromatid cohesion103,104,124. Pericentric heterochromatin remains condensed throughout the cell cycle and individual pericentromeres come together into large clusters called chromocentres124, as shown by DNA fluorescence in situ hybridization (FISH) for pericentric satellite repeats in mouse embryonic fibroblasts (see the figure, part b). At the molecular level, pericentric heterochromatin is characterized by extensive DNA methylation and specific histone methylation marks, such as dimethylated and trimethylated H3K9 (H3K9me2 and H3K9me3, respectively), that are bound by heterochromatin protein 1 (HP1; see the figure, part c). There are three HP1 proteins in mammals: HP1α, HP1β and HP1γ (also known as CBX5, CBX1 and CBX3, respectively). RNA interference (RNAi) contributes to heterochromatin integrity in fission yeast and plants172; however, a direct connection in flies and mammalian cells is so far lacking. Not every epigenetic mark is present at pericentric heterochromatin in all model organisms. Scale bar, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole. REVIEWS 194 | mARcH 2009 | VOlume 10 www.nature.com/reviews/molcellbio © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS Fork direction ork direction Lagging DNM O General O DNA methylation dependen Domain HPL-SUV39HI O DNA methylation independent specific New nucleosomes Figure 1 Asymmetric DNA replication and coupling of inheritance of DNA and histone marks a An intrinsic strand bias at DNA replication. DNA replication occurs in the 5 to 3 direction. One strand is replicated as the leading strand and the other as the lagging strand is b Proliferating cell nuclear antigen(PCNA) molecules associate with the 3'end of newly synthesized DNA. This results in the loading of PCNA on to the two strands. c Maintenance of dNa and specific histone modifications at the replication fork Homotrimeric PCNA recruits general factors that function at all PR-SET7 and SETD8)y8-75, chromatin remodellers(Williams syndrome transcription factor(STF-SNF2H (als ko,, forks, such as histone modifiers(histone deacetylases(HDACs) and the Lys methyltransferase SET8(also known as SMARCAS)) and chromatin assembly factor 1(CAF1; also known as CHAF1) Depending on the presence of DNA methylation, PCNA together with NP95(also called UHRF1 and ICBP90)recruits DNA methyltransferase 1(DNMT1). which methylates hemimethylated CpG n daughter strands 32.33. Certain histone modifiers use the dNA methylation machinery as a template-for example, HDAC activity is recruited by DNMTl and NP95(REFS 81, 82), and DNMT1 interacts with the Lys methyltransferase G9a(also known as KMT1C)In DNA methylation-rich regions, CAF1 forms a complex with methyl CpG-binding protein 1(MBD1)and the Lys methyltransferase SETDB1(also known as KMT1E), thereby coupling histone deposition with histone methylation CAFl also contributes to the maintenance of heterochromatin protein 1(HP1)in a DNA-methylation-independent process 10.HPl, in turn, interacts with the histone methyltransferase SUV39H1(also known as KMT1A ith high fidelity. The maintenance of DNA methyl- NP95 binds preferentially to hemimethylated DNA-36 ation at the fork is ensured by DNA methyltransferase 1 interacts with DNMTI and is required for its localization (NMTI), owing to its affinity for hemimethylated dNA to replicating heterochromatic regions(FIG. 1c). Indeed, in vitro242 and its interaction with PCNA". However, the deletion of NP95 results in methylation defectsthat mechanism by which methylation maintenance is ensured resemble those that are observed following the loss of in a faithful manner was unclear, as DNMTI also shows DNMTI(REF. 37), which suggests that NP95 has a domi de novo methylation activity and its ability to bind nant role in tethering maintenance methyltransferase PCNA is not absolutely required for DNA methylation activity to newly replicated DNA. The maintenance of maintenance. Recent evidence now suggests that the DNA methylation further requires the ATP-dependent DNA methyltransferase SET-and RING-associated(SRA)-domain-containing chromatin-remodelling factor decreased DNA methy ne that transfers proteins variant in methylation 1(VIMI)in Arabidopsis ation 1 (DDMI)in A thaliana and LSH (also known ethyl groups fro thaliana and NP95(also called UHRFI and ICBP90) as HELLS)in mice, which have been suggested specific adenines or cytosines in mammals constitute an additional mechanistic link provide access of the methylation machinery to newly between hemimethylated DNA and DNMTI (REFS 30-33). replicated DNA NATURE REVIEWS MOLECULAR CELL BIOLOGY 22009 Macmillan Publishers Limited All rights reserved

DNMT1 NP95 Nature Reviews | Molecular Cell Biology 5′ 5′ 3′ 5′ 3′ 5′ 3′ 5′ 3′ 3′ 5′ 3′ a b Leading c Lagging Leading Lagging PCNA PCNA Fork direction Fork direction HDAC SET8 HDAC WSTF– SNF2H MBD1–SETDB1 HP1–SUV39H1 HDAC G9a CAF1 General DNA methylation dependent DNA methylation independent DNA methylation Domain specific Parental nucleosomes New nucleosomes Replication machinery DNA methyltransferase An enzyme that transfers methyl groups from S-adenosylmethionine to specific adenines or cytosines in DNA. with high fidelity. The maintenance of DNA methyl￾ation at the fork is ensured by DNA methyltransferase 1 (DNmT1), owing to its affinity for hemimethylated DNA in vitro24,25 and its interaction with PcNA26. However, the mechanism by which methylation maintenance is ensured in a faithful manner was unclear, as DNmT1 also shows de novo methylation activity27 and its ability to bind PcNA is not absolutely required for DNA methylation maintenance28,29. Recent evidence now suggests that the SeT­ and RING­associated (SRA)­domain­containing proteins variant in methylation 1 (VIm1) in Arabidopsis thaliana and NP95 (also called uHRF1 and IcBP90) in mammals constitute an additional mechanistic link between hemimethylated DNA and DNmT1 (ReFs 30–33). NP95 binds preferentially to hemimethylated DNA34–36, interacts with DNmT1 and is required for its localization to replicating heterochromatic regions32 (FIG. 1c). Indeed, deletion of NP95 results in methylation defects33 that resemble those that are observed following the loss of DNmT1 (ReF. 37), which suggests that NP95 has a domi￾nant role in tethering maintenance methyltransferase activity to newly replicated DNA. The maintenance of DNA methylation further requires the ATP­dependent chromatin­remodelling factor decreased DNA methyl￾ation 1 (DDm1) in A. thaliana38,39 and lSH (also known as HellS) in mice40, which have been suggested to provide access of the methylation machinery to newly replicated DNA38. Figure 1 | asymmetric DNa replication and coupling of inheritance of DNa and histone marks. a | An intrinsic strand bias at DNA replication. DNA replication occurs in the 5′ to 3′ direction. One strand is replicated as the leading strand and the other as the lagging strand18. b | Proliferating cell nuclear antigen (PCNA) molecules associate with the 3′ end of newly synthesized DNA. This results in the loading of PCNA on to the two strands. c | Maintenance of DNA and specific histone modifications at the replication fork. Homotrimeric PCNA recruits general factors that function at all forks, such as histone modifiers (histone deacetylases (HDACs) and the Lys methyltransferase SET8 (also known as KMT5A, PR-SET7 and SETD8))73–75, chromatin remodellers (Williams syndrome transcription factor (WSTF)–SNF2H (also known as SMARCA5))76 and chromatin assembly factor 1 (CAF1; also known as CHAF1)21. Depending on the presence of DNA methylation, PCNA together with NP95 (also called UHRF1 and ICBP90) recruits DNA methyltransferase 1 (DNMT1), which methylates hemimethylated CpG sites on daughter strands26,32,33. Certain histone modifiers use the DNA methylation machinery as a template — for example, HDAC activity is recruited by DNMT1 and NP95 (ReFs 81,82), and DNMT1 interacts with the Lys methyltransferase G9a (also known as KMT1C)83. In DNA methylation-rich regions, CAF1 forms a complex with methyl CpG-binding protein 1 (MBD1) and the Lys methyltransferase SETDB1 (also known as KMT1E), thereby coupling histone deposition with histone methylation79,80. CAF1 also contributes to the maintenance of heterochromatin protein 1 (HP1) in a DNA-methylation-independent process128,130. HP1, in turn, interacts with the histone methyltransferase SUV39H1 (also known as KMT1A)68. REVIEWS NATuRe ReVIeWS | Molecular cell Biology VOlume 10 | mARcH 2009 | 195 © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS fide epigenetic mark. Although we have learnt about the dimers maintenance mechanisms that ensure the stable propa gation of marks, it will also be important to consider mechanisms that enable the removal of these marks NAPL FACT ones to fully comprehend the dynamic behaviour of dnA ethylation, as suggested by recent reports I B+4 Inheritance of histones and their modifications? DNA and its methylation marks are replicated using semi- conservative mechanisms of inheritance, in which information is copied from a template. Passage of the Parental histone New histones replication fork also disrupts parental nucleosomes that carry post-translational modifications. In order to be 感 heritable and therefore to qualify as epigenetic marks, these histones and their modifications must be correctly yo synthesized reassembled behind the fork However, an obvious tem- plate for nucleosome reassembly is lacking. Given that DNA tside of S phase the exchange of the replicative histone H3 variant H3. 1 and histone H4 is minimal compared H3-H4 cytoplasm with the rapid exchange of H2A and H2B5,46,H3 and H4, along with their associated marks, have arisen as likely candidates to transmit information from one cell ycle to the next. Therefore, to avoid the loss of infor mation that is encoded in histone modifications, proper coordination is required between the recycling of paren tal H3-H4 dimers with their histone marks, along with Nucleosome assembly involves the deposition of one H3-H4), tetramer, which can exist in an intermedi ate H3-H4 dimeric form, onto DNA, followed by the Only old Chromatin deposition of two H2A-H2B dimers"(FIG2a).Histone aperones have key roles as histone acceptors and donors that assist in the disruption and reassembly of New H3-H4 dimer nucleosomes. They control histone provision locally 9 Parental mark nd exhibit specificity for particular histones or even a P New mark specific histone variant". Importantly, the H3. 1-H4 haperone chromatin assembly factor 1(CAFl; also Figure 2 Nucleosome dynamics and mixing of parental and new H3-H4 dimers. al The incorporation of histone(H3-H4), tetramers onto DNA, followed by the addition of Known as CHAFl)is recruited to the replication fork two histone H2A-H2 B dimers to form a nucleosome core particle. Prior to deposition, through an interaction with PCNA along with other his- H3-H4 and H2A-H2B exist as dimers that are complexed to specific histone chaperones. tone modifiers, such as histone deacetylases(hdacs)and blOn chromatin disruption at replication, parental H3-H4 tetramers with histone marks Lys methyltransferases( see below). CAFl is composed ith the chaperone anti-silencing function 1(ASF1)263 Nucleosomes with only old H3-H4 nate nucleosome assembly during DNA replication"or re formed when unsplit parental tetramers are transferred directly onto daughter strands at sites of DNA repair- by facilitating the deposition orwhen two parental H3-H4 dimers reassociate. Newly synthesized H3-H4 dimers with of newly synthesized H3. 1-H4(REF. 52) their typical marks are complexed with the chaperones ASF1 and chromatin assembly Another H3-H4 chaperone, ASFI, interacts directly factor 1(CAF1; also known as CHAF1) Nucleosomes might be formed on the daughter with the CAFl p60 subunit and functions synergist strands from one parental and one new H3-H4 dimer (indicated as mixed)or exclusively cally with CAFl in DNA Synthesis-dependent chromatin from two new H3-H4 dimers (indicated as only new). Nucleosomes that contain mixed nd new histones undergo maturation after formation. FACT, facilitates chromatin assembly by acting as a donor of newly synthesized ranscription: HIRA, Hir-related protein A: NAPl, nucleosome assembly protein 1. histones. Furthermore, ASFI is directly linked to the replication fork machinery through interactions with components of the putative replicative helicase DNA methylation patterns can be reproduced faith- Downregulation of ASFI slows down S-phase progres ully after the passage of the replication fork by taking sion and impairs DNA unwinding because of defects in advantage of a combination of factors: semi-conservative histone dynamics. The newly synthesized histones that replication, which gives rise to hemimethylated DNA; are associated with chaperones, such as CAFl and ASFI the recognition of the hemimethylated daughter strand carry the evolutionarily conserved combination of the by NP95; and the association of DNMTI with the rep- K5 and K12 acetylation marks on H4(REFS 54, 55), which lication machinery. These mechanisms ensure a stable are associated with the deposition of new histones and are propagation of DNA methylation patterns and reinforce removed during chromatin maturation In budding yeast, the view that DNA methylation is a prototype of a bona new H3 is acetylated at residue K56( H3K56ac), which 22009 Macmillan Publishers Limited All rights reserved

CAF1 Nature Reviews | Molecular Cell Biology DNA H2A–H2B dimers H2A–H2B dimers H3–H4 tetramer + + Histone chaperones NAP1, FACT Histone chaperones ASF1, CAF1, HIRA a b Parental histones New histones Chromatin H3–H4 tetramer ASF1 ASF1 ASF1 ASF1 ASF1 DNA H3–H4 dimers Reassociation Nucleus Cytoplasm Chromatin Maturation Maturation De novo synthesized H3–H4 dimers Only old Mixed Only new ‘Unsplit’ ‘Split’ Parental mark Old H3–H4 dimer New mark New H3–H4 dimer H3–H4 dimers DNA methylation patterns can be reproduced faith￾fully after the passage of the replication fork by taking advantage of a combination of factors: semi­conservative replication, which gives rise to hemimethylated DNA; the recognition of the hemimethylated daughter strand by NP95; and the association of DNmT1 with the rep￾lication machinery. These mechanisms ensure a stable propagation of DNA methylation patterns and reinforce the view that DNA methylation is a prototype of a bona fide epigenetic mark. Although we have learnt about the maintenance mechanisms that ensure the stable propa￾gation of marks, it will also be important to consider mechanisms that enable the removal of these marks to fully comprehend the dynamic behaviour of DNA methylation, as suggested by recent reports41–43. Inheritance of histones and their modifications? DNA and its methylation marks are replicated using semi￾conservative mechanisms of inheritance, in which information is copied from a template44. Passage of the replication fork also disrupts parental nucleosomes that carry post­translational modifications. In order to be heritable and therefore to qualify as epigenetic marks, these histones and their modifications must be correctly reassembled behind the fork13. However, an obvious tem￾plate for nucleosome reassembly is lacking. Given that outside of S phase the exchange of the replicative histone H3 variant H3.1 and histone H4 is minimal compared with the rapid exchange of H2A and H2B45,46, H3 and H4, along with their associated marks, have arisen as likely candidates to transmit information from one cell cycle to the next. Therefore, to avoid the loss of infor￾mation that is encoded in histone modifications, proper coordination is required between the recycling of paren￾tal H3–H4 dimers with their histone marks, along with the incorporation of newly synthesized histones13. Nucleosome assembly involves the deposition of one (H3–H4)2 tetramer, which can exist in an intermedi￾ate H3–H4 dimeric form, onto DNA, followed by the deposition of two H2A–H2B dimers47 (FIG. 2a). Histone chaperones have key roles as histone acceptors and donors that assist in the disruption and reassembly of nucleosomes. They control histone provision locally and exhibit specificity for particular histones or even a specific histone variant48. Importantly, the H3.1–H4 chaperone chromatin assembly factor 1 (cAF1; also known as cHAF1) is recruited to the replication fork through an interaction with PcNA along with other his￾tone modifiers, such as histone deacetylases (HDAcs) and Lys methyltransferases19,21 (see below). cAF1 is composed of three subunits — p150, p60 and p48 — that coordi￾nate nucleosome assembly during DNA replication49,50 or at sites of DNA repair22,51 by facilitating the deposition of newly synthesized H3.1–H4 (ReF. 52). Another H3–H4 chaperone, ASF1, interacts directly with the cAF1 p60 subunit53 and functions synergisti￾cally with cAF1 in DNA synthesis­dependent chromatin assembly by acting as a donor of newly synthesized histones. Furthermore, ASF1 is directly linked to the replication fork machinery through interactions with components of the putative replicative helicase23. Downregulation of ASF1 slows down S­phase progres￾sion and impairs DNA unwinding because of defects in histone dynamics23. The newly synthesized histones that are associated with chaperones, such as cAF1 and ASF1, carry the evolutionarily conserved combination of the K5 and K12 acetylation marks on H4 (ReFs 54,55), which are associated with the deposition of new histones and are removed during chromatin maturation. In budding yeast, new H3 is acetylated at residue K56 (H3K56ac), which Figure 2 | Nucleosome dynamics and mixing of parental and new H3–H4 dimers. a | The incorporation of histone (H3–H4)2 tetramers onto DNA, followed by the addition of two histone H2A–H2B dimers to form a nucleosome core particle. Prior to deposition, H3–H4 and H2A–H2B exist as dimers that are complexed to specific histone chaperones. b | On chromatin disruption at replication, parental H3–H4 tetramers with histone marks can either be preserved (unsplit) or broken up into dimers (split), potentially by interacting with the chaperone anti-silencing function 1 (ASF1)62,63. Nucleosomes with only old H3–H4 are formed when unsplit parental tetramers are transferred directly onto daughter strands or when two parental H3–H4 dimers reassociate. Newly synthesized H3–H4 dimers with their typical marks are complexed with the chaperones ASF1 and chromatin assembly factor 1 (CAF1; also known as CHAF1)59. Nucleosomes might be formed on the daughter strands from one parental and one new H3–H4 dimer (indicated as mixed) or exclusively from two new H3–H4 dimers (indicated as only new). Nucleosomes that contain mixed and new histones undergo maturation after formation. FACT, facilitates chromatin transcription; HIRA, Hir-related protein A; NAP1, nucleosome assembly protein 1. REVIEWS 196 | mARcH 2009 | VOlume 10 www.nature.com/reviews/molcellbio © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS CAFI and the yeast-specific histone chaperon (REFS 56, 57). Whereas the presence of the H3K56 e(using neighbouring has been reported in humans, its abundance seems mark as template) OR dilution (not shown) limited and its association with new histone deposi- tion is not documented. Furthermore, homologues of 感感 Rtt106 and the Lys acetyltransferase Rtt109(also known as Katl1), which acts on H3K56, have yet to be identi fied in humans. So, whether H3K56ac or an unidentified modification have similar roles in mammals remains to be investigated. Notably, newly synthesized histones H3 and H4 are present as dimers in pre-deposition complexes with histone chaperones(FIG 2a). Although this principle is clearly established for newly synthesized histones, the fate of parental H3-H4 histone dimers that are thought to be deposited as tetramers on the daughter strands might also have to be reconsidered. The fact that histones Maintenance by default emimodified nucleosome used H3 and H4 exist as stable tetramers in solution in the as a template for further modification) absence of DNA argues against the existence of parental H3-H4 dimers. However, structural data now show that HDAC the association of ASFl, and potentially also p48 and p55, with histones is incompatible with a tetrameric rrying parental marks can be detected in association with asl under conditions in which the helicase and le polymerase are uncoupledsupports the hypothesis that ASFI is involved in tetramer splitting and that it functions as an acceptor of recycled parental dimers Therefore, it is indeed possible that parental tetramers (with their own marks) are split and redistribute onto Maintenance requires interstrand daughter strands as dimers. This affects histone dynamic crosstalk OR switch (not shown at the fork and might produce either mixed tetramers that comprise parental and new dimers(FIG. 2b), or nd nucleosomes that comprise only old histones if paren tal dimers reassociate. This second scenario requires either that the old dimers are held in close contact or away from the new ones, or that some recognition event ensures that the correct old dimers are brought back together in the same particle. The spatial organization of DNA at the fork might facilitate these mechanisms 9 Parental mark C Old H3-H4 dime If modifications on the new histones are guided by New H3-H4 dimer modifications of parental histones, the way in which Figure 3 Fate of old and new H3-H4 dimers and their marks at the fork. Three parental histones are distributed to the daughter strands ill determine the degree of conservation of histone parental mark is recognized by a chromatin-binding protein, or reader protein, that in turn marks. Current models suggest that the distribution recruits a chromatin modifier, or writer protein a Random histone distribution. Parental of both parental and newly synthesized histones onto histone H3 and H4 with marks(unsplit or reassociated dimers) are distributed randomly daughter strands occurs in a random fashion(FIG 3a). To onto daughter strands and chromatin density is restored by the deposition of new H3-H4 avoid the dilution of histone marks, the maintenance of dimers. To avoid the dilution of histone marks, active maintenance requires first a modifications could be achieved by using a neighbour deacetylation step, which involves a histone deacetylase(HDAC), followed by histone ing histone as a template. A possible mechanism could modification that is guided by neighbouring parental nucleosomes(an interparticle be envisaged in which the parental mark is recognized process).b|Semi-conservative histone distribution. Parental dimers with marks segregate by a chromatin-binding protein, or reader protein, that evenly onto each daughter strand and nucleosomes are completed by the deposition of in turn recruits a chromatin modifier, or writer protein new H3-H4 dimers. After deacetylation, hemimodified' nucleosomes provide a template This has been suggested for the self-reinforcing loop for the transmission of parental marks to newly deposited H3-H4 dimers (an intraparticle in the maintenance of heterochromatin protein 1(HPI process). c Asymmetric histone distribution. Parental H3-H4 dimers with marks are redistributed onto daughter strands in an asymmetric manner. This is possibly dictated by at pericentric heterochromatinb7-(see below). Such a the intrinsic strand bias that is introduced during DNA replication, and induces a switch mechanism probably operates in repetitive regions one chromatin state to another. The maintenance of histone modifications requires in which long arrays of nucleosomes carry the same strand crosstalk. arks, but cannot apply to regions in which particular NATURE REVIEWS MOLECULAR CELL BIOLOGY 22009 Macmillan Publishers Limited All rights reserved

Nature Reviews | Molecular Cell Biology a Random Distribution Consequence c Asymmetric b Semi-conservative Maintenance (using neighbouring mark as template) OR dilution (not shown) Maintenance by default (hemimodified nucleosome used as a template for further modification) HDAC HDAC Maintenance requires interstrand crosstalk OR switch (not shown) R W R W Reader Writer HDAC Parental mark New mark R W Old H3–H4 dimer New H3–H4 dimer ‘Interparticle’ ‘Intraparticle’ ‘Interstrand’ HDAC HDAC regulates the nucleosome assembly that is dependent on cAF1 and the yeast­specific histone chaperone Rtt106 (ReFs 56,57). Whereas the presence of the H3K56ac mark has been reported in humans58, its abundance seems limited and its association with new histone deposi￾tion is not documented. Furthermore, homologues of Rtt106 and the Lys acetyltransferase Rtt109 (also known as Kat11), which acts on H3K56, have yet to be identi￾fied in humans. So, whether H3K56ac or an unidentified modification have similar roles in mammals remains to be investigated. Notably, newly synthesized histones H3 and H4 are present as dimers in pre­deposition complexes with histone chaperones59 (FIG. 2a). Although this principle is clearly established for newly synthesized histones, the fate of parental H3–H4 histone dimers that are thought to be deposited as tetramers on the daughter strands might also have to be reconsidered. The fact that histones H3 and H4 exist as stable tetramers in solution in the absence of DNA60 argues against the existence of parental H3–H4 dimers. However, structural data now show that the association of ASF1, and potentially also p48 and p55, with histones is incompatible with a tetrameric structure61–65. In addition, the fact that some histones carrying parental marks can be detected in association with ASF1 under conditions in which the helicase and the polymerase are uncoupled23 supports the hypothesis that ASF1 is involved in tetramer splitting and that it functions as an acceptor of recycled parental dimers. Therefore, it is indeed possible that parental tetramers (with their own marks) are split and redistribute onto daughter strands as dimers. This affects histone dynamics at the fork and might produce either mixed tetramers that comprise parental and new dimers (FIG. 2b), or nucleosomes that comprise only old histones if paren￾tal dimers reassociate. This second scenario requires either that the old dimers are held in close contact or away from the new ones, or that some recognition event ensures that the correct old dimers are brought back together in the same particle. The spatial organization of DNA at the fork might facilitate these mechanisms (FIG. 1a). If modifications on the new histones are guided by modifications of parental histones, the way in which parental histones are distributed to the daughter strands will determine the degree of conservation of histone marks. current models suggest that the distribution of both parental and newly synthesized histones onto daughter strands occurs in a random fashion (FIG. 3a). To avoid the dilution of histone marks, the maintenance of modifications could be achieved by using a neighbour￾ing histone as a template. A possible mechanism could be envisaged in which the parental mark is recognized by a chromatin­binding protein, or reader protein66, that in turn recruits a chromatin modifier, or writer protein. This has been suggested for the self­reinforcing loop in the maintenance of heterochromatin protein 1 (HP1) at pericentric heterochromatin67–70 (see below). Such a mechanism probably operates in repetitive regions in which long arrays of nucleosomes carry the same marks, but cannot apply to regions in which particular Figure 3 | Fate of old and new H3–H4 dimers and their marks at the fork. Three possibilities for the distribution of parental histones are presented. In each case, the parental mark is recognized by a chromatin-binding protein, or reader protein, that in turn recruits a chromatin modifier, or writer protein. a | Random histone distribution. Parental histone H3 and H4 with marks (unsplit or reassociated dimers) are distributed randomly onto daughter strands and chromatin density is restored by the deposition of new H3–H4 dimers. To avoid the dilution of histone marks, active maintenance requires first a deacetylation step, which involves a histone deacetylase (HDAC), followed by histone modification that is guided by neighbouring parental nucleosomes (an interparticle process). b | Semi-conservative histone distribution. Parental dimers with marks segregate evenly onto each daughter strand and nucleosomes are completed by the deposition of new H3–H4 dimers. After deacetylation, ‘hemimodified’ nucleosomes provide a template for the transmission of parental marks to newly deposited H3–H4 dimers (an intraparticle process). c | Asymmetric histone distribution. Parental H3–H4 dimers with marks are redistributed onto daughter strands in an asymmetric manner. This is possibly dictated by the intrinsic strand bias that is introduced during DNA replication, and induces a switch from one chromatin state to another. The maintenance of histone modifications requires interstrand crosstalk. REVIEWS NATuRe ReVIeWS | Molecular cell Biology VOlume 10 | mARcH 2009 | 197 © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS Box 3 Spatial and temporal regulation of replication istone dimers. A third possibility, which might occur at certain times and in certain chromatin domains, is that parental and new histones segregate asymmetrically This might be dictated by the intrinsic strand bias that is introduced at DNA replication, as discussed above (FIGS 1b,3c]. This might help to induce a switch by pro- viding a blank template to allow a change in cell fate on one of the two daughter strands. To faithfully copy infor mation from parental to new nucleosomes, interstrand osstalk would be required. Although evidence for this kind of mechanism is currently lacking, it is possible to ge a folding in space that brings the two daughter DAP strands into close proximity. In summary, several models that are not necessarily mutually exclusive have been proposed to describe how new and recycled histones are incorporated and modi ontexts(for example, different cell types and particular subdomains of the nucleus )in order to evaluate the effect of histone dynamics at the replication fork on the stability Euchromatic regions d plasticity of an epigenetic state. n the nucleus, distinct chromatin domains occupy different compartments and replicate Connecting inheritance of DNA and histone marks at different times, with the classic example of heterochromatin usually replicating late In the reader-writer model for the inheritance ofhistone nd euchromatin early".Such a temporal and spatial replication programme highlights marks (FIG. 3), marks on neighbouring parental nucleo- the capacity of the cell to distinguish one domain from another. Characteristic patterns somes serve as a template for modifications of newly different times during S phase/47s. In mice, at least three s-phase patterns that incorporated histones. This maturation step might take ACcupy different subnuclear compartments can be distinguished 8-early, mid and late place at later stages in the cell cycle. However,marks phase--as shown by pulse labelling with a nucleotide analogue(see the figure). ould also be imposed in a replication-coupled manner and might be coordinated with the timing of domain domains remain visible by 4, 6-diamidino-2-phenylindole(DAPI)staining, and replication (BOX 3). For replication-coupled mainte- bromodeoxyuridine( BrdU)incorporation is detected at the periphery of the domain, nance, two situations can be considered: first, common thereby revealing a specific organization. Replication timing patterns are also particular factors at all replication forks can affect marking; and to the differentiation state of a cell. Such a spatial and temporal organization could be second, domain-specific factors are modulated by the exploited to self-maintain or propagate domains by facilitating the packaging of DNA local chromatin environment and by pre-existing marks, into diferent types of chromatin during S phase, depending on when and where it is such as DNA methylation (FIG. Ic) and/or by using specialized replication mechanisms, Proteins that are targeted to early or landing pad for different chromatin modifiers z pcna ecular terms to provide specificity Histone deacetylase 2(HDAC2)2, methyl ding protein 2(MBD2HMBD3 (REF 179)and williams syndrome transcription (also known as KMT5A, PR-SET7 and SETD8), which is factor(WSTFH-SNFZHsO are examples of factors that are reported to be specific to implicated in monomethylation of H4K20(REFS 74, 75)as late replication foci. a current challenge is to understand how spatial and temporal well as chromatin remodelling activity. PCNA, together organization of replication is established and to solve the long-standing issue of the with CAFl, remains on replicated DNA for-20 min".8 itation of replication origins in mammals. Scale bar, 5 um. During this time window, newly replicated chrom itin undergoes modifications, including the removal marks are restricted to only one or two nucleosomes. of acetylation marks on residues 5 and K12 of newly A similar self-maintaining process has now been pro- incorporated histone H4(REFS 54,55]. posed for the maintenance of the repressive methyl- At DNA methylation-rich regions, pre-existing meth- tion mark H3K27me3(H3 trimethylated on residue ylation and its associated maintenance machinery could K27)during replication, in which polycomb repressive guide the placement of histone modifications. In those omplex 2(PRC2)-which is responsible for setting regions, methyl CpG-binding protein 1(MBDI), which the H3K27me3 mark binds to its own methylation is found in a complex with the Lys methyltransferase ant site. It will be important to determine when the par- SETDB1 (also known as KMTiE) can interact with is expressed and incorporated ental marks are actually imposed after the passage of the CAFI during replication which suggests that there is a ample, H3.1 and H3.2). replication fork to evaluate how tightly the inheritance is connection between histone deposition and the setting of iant coupled to replication. modifications Reported interactions of the DNA methyl Split parental tetramers could also distribute in a semi- transferase enzyme DNMTi with the histone-modifying cell cycle and is incorporated in conservative manner(FIG. 3b). As in the hemimethyla enzymes HDACI (REF. 81), HDAC2 (REF. 82) and the Ly manner (for example, H3.3 and DNA Scenario above,hemimodified'nucleosomes methyltransferase G9a(also known as KMTIC)a),might the centromere-specific provide a template that instructs the appropriate choice ensure a coordination between the imposition of marks 13). of modification to impose on newly deposited H3-H4 on DNA and histones 198 MARCH 2009 VOLUME 10 22009 Macmillan Publishers Limited All rights reserved

Nature Reviews | Molecular Cell Biology S phase: Early Mid Late ‘Euchromatic regions’ ‘Heterochromatic regions’ DAPI BrdU Histone H3 variant A replicative histone H3 variant is expressed and incorporated during DNA replication (for example, H3.1 and H3.2), whereas a replacement variant is expressed throughout the cell cycle and is incorporated in a DNA-synthesis-independent manner (for example, H3.3 and the centromere-specific histone H3 variant CenH3). marks are restricted to only one or two nucleosomes. A similar self­maintaining process has now been pro￾posed for the maintenance of the repressive methyl￾ation mark H3K27me3 (H3 trimethylated on residue K27) during replication, in which polycomb repressive complex 2 (PRc2) — which is responsible for setting the H3K27me3 mark — binds to its own methylation site71. It will be important to determine when the par￾ental marks are actually imposed after the passage of the replication fork to evaluate how tightly the inheritance is coupled to replication. Split parental tetramers could also distribute in a semi￾conservative manner (FIG. 3b). As in the hemimethylated DNA scenario above, ‘hemimodified’ nucleosomes provide a template that instructs the appropriate choice of modification to impose on newly deposited H3–H4 histone dimers. A third possibility, which might occur at certain times and in certain chromatin domains, is that parental and new histones segregate asymmetrically72. This might be dictated by the intrinsic strand bias that is introduced at DNA replication, as discussed above (FIGs 1b,3c). This might help to induce a switch by pro￾viding a blank template to allow a change in cell fate on one of the two daughter strands. To faithfully copy infor￾mation from parental to new nucleosomes, interstrand crosstalk would be required. Although evidence for this kind of mechanism is currently lacking, it is possible to envisage a folding in space that brings the two daughter strands into close proximity. In summary, several models that are not necessarily mutually exclusive have been proposed to describe how new and recycled histones are incorporated and modi￾fied. It will be important to assess these models in different contexts (for example, different cell types and particular subdomains of the nucleus) in order to evaluate the effect of histone dynamics at the replication fork on the stability and plasticity of an epigenetic state. Connecting inheritance of Dna and histone marks In the reader–writer model for the inheritance of histone marks (FIG. 3), marks on neighbouring parental nucleo￾somes serve as a template for modifications of newly incorporated histones. This maturation step might take place at later stages in the cell cycle. However, marks could also be imposed in a replication­coupled manner and might be coordinated with the timing of domain replication (BOX 3). For replication­coupled mainte￾nance, two situations can be considered: first, common factors at all replication forks can affect marking; and second, domain­specific factors are modulated by the local chromatin environment and by pre­existing marks, such as DNA methylation (FIG. 1c). PcNA on all replication forks can function as a landing pad for different chromatin modifiers17. PcNA recruits HDAcs73 and the lys methyltransferase SeT8 (also known as KmT5A, PR­SeT7 and SeTD8), which is implicated in monomethylation of H4K20 (ReFs 74,75) as well as chromatin remodelling activity76. PcNA, together with cAF1, remains on replicated DNA for ~20 min77,78. During this time window, newly replicated chrom￾atin undergoes modifications, including the removal of acetylation marks on residues K5 and K12 of newly incorporated histone H4 (ReFs 54,55). At DNA methylation­rich regions, pre­existing meth￾ylation and its associated maintenance machinery could guide the placement of histone modifications. In those regions, methyl cpG­binding protein 1 (mBD1), which is found in a complex with the lys methyltransferase SeTDB1 (also known as KmT1e)79, can interact with cAF1 during replication80, which suggests that there is a connection between histone deposition and the setting of modifications. Reported interactions of the DNA methyl￾transferase enzyme DNmT1 with the histone­modifying enzymes HDAc1 (ReF. 81), HDAc2 (ReF. 82) and the lys methyltransferase G9a (also known as KmT1c)83, might ensure a coordination between the imposition of marks on DNA and histones. Box 3 | spatial and temporal regulation of replication In the nucleus, distinct chromatin domains occupy different compartments and replicate at different times, with the classic example of heterochromatin usually replicating late and euchromatin early173. Such a temporal and spatial replication programme highlights the capacity of the cell to distinguish one domain from another. Characteristic patterns of replication can be visualized in cells that are labelled with nucleotide analogues at different times during S phase174,175. In mice, at least three S-phase patterns that occupy different subnuclear compartments can be distinguished128 — early, mid and late S phase — as shown by pulse labelling with a nucleotide analogue (see the figure). Note that when pericentric heterochromatin replicates in mid S phase, pericentric domains remain visible by 4′,6-diamidino-2-phenylindole (DAPI) staining, and bromodeoxyuridine (BrdU) incorporation is detected at the periphery of the domain, thereby revealing a specific organization. Replication timing patterns are also particular to the differentiation state of a cell176. Such a spatial and temporal organization could be exploited to self-maintain or propagate domains by facilitating the packaging of DNA into different types of chromatin during S phase, depending on when and where it is replicated173,177. This could be achieved by using local concentrations of specific factors178 and/or by using specialized replication mechanisms. Proteins that are targeted to early or late replication forks could be instrumental in revealing how these features can translate into molecular terms to provide specificity. Histone deacetylase 2 (HDAC2)82, methyl CpG-binding protein 2 (MBD2)–MBD3 (ReF. 179) and Williams syndrome transcription factor (WSTF)–SNF2H180 are examples of factors that are reported to be specific to late replication foci. A current challenge is to understand how spatial and temporal organization of replication is established and to solve the long-standing issue of the initiation of replication origins in mammals. Scale bar, 5 μm. REVIEWS 198 | mARcH 2009 | VOlume 10 www.nature.com/reviews/molcellbio © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS Particular features that are created at the time of It is possible that the dilution of H3.3 and its marks DNA replication in a particular domain might also be by one-half after one cell cycle might not affect the exploited. NP95 has affinity for both hemimethylated transcriptional readout of a region. Thus, sustained DNA and histones-b64, and specifically interacts with active gene expression, combined with modifications on peptides that are methylated at H3K9 in vitro by poten- parental H3. 3, might recruit factors that modify newly tially reading histone marks. In addition, NP95 was incorporated H3. 1 with the appropriate marks, and found in a complex with HDACs and G9a"12. Therefore, H3. 3 incorporation might be stimulated. Consistent with as well as binding to hemimethylated DNA, NP95 could this hypothesis, arrays of nucleosomes that contain both interpret the histone environment, thereby creating a H3. 3 and H3. 1 nucleosomes have been observed, and feedback mechanism that involves the mutual reinforce- analysis of histone modifications in this context show ment of histone and DNA methylation marks. In this that, when adjacent to H3. 3 nucleosomes, H3. 1 nucleo- situation histone marks would influence the inheritance somes accumulate active marks However, dilution of of DNA methylation. Further chromatin-binding pro- H3. 3 over a number of generations might be reconciled teins or chromatin modifiers with dual affinity for both by the replication-independent incorporation of H3.3 DNA methylation and a particular histone modification that is promoted by chaperones, such as Hir-related are likely to be identified. protein A(HIRA), following transcription(FIG 4a) The examples above show how histone and DNA methylation in a repressive domain could be maintained Inheritance of CENP-A. The histone H3 variant CENP-A at the replication fork. However, how active chromatin marks the site of centromere identity 49. The associa- marks are propagated is less clear. Recently, transmission tion of CENP-A with centromeres is extremely stable, as of an active state through nuclear transfer in Xenopus shown by quantitative fluorescence recovery after photo- laevis has been reported, and it has been proposed that bleaching(FRAP)analysis, and it remains associated the replacement histone variant H3.3 is required for epi- through cell division%. Although the exact mechanism of genetic memory 6. To evaluate this hypothesis, it is nec- CENP-A deposition at centromeres remains enigmatic, it essary to better understand the mechanisms that involve is a replication-independent process, as is the deposition replication-independent histone exchange processes and of H3.3 CENP-A deposition was first proposed the replacement of histone variants. to occur in G2 phase, because CENP-A assembly can take place in the presence of the DNA replication inhibitor Inheritance of histone variants outside S phase aphidicolin, and CENPA mRNA and the CENP-a pro Histone variants can mark a particular chromatin state: tein peak in G2 phase 7. Recent evidence in mammalian H3. 3 is enriched at active regions, whereas the unique cells now suggests that the loading of new CENP-Aonto incorporation of the centromere-specific histone H3 centromeres is restricted to a discrete cell cycle window in An enzyme that removes acetyl variant CenH3(CENP-A in humans) specifies the site of late telophase-early Gl phase, but the mechanism and groups from histones. centromere identity. Together with the replicative vari- the specific chaperone that facilitate CENP-A deposition ants H3. 1 and H3. 2, the replacement variants H3.3 and remain to be deciphered. n enzyme that catalyses the CENP-A constitute the major histone H3 isotypes that Centromeric DNA is replicated during S phase, in addition of a methyl group to are known in mammals. During S phase, H3. I and which parental CENP-A nucleosomes are distributed to pecific Lys residues in histones H3. 2 are exclusively incorporated, whereas the deposi- daughter strands 7. 9. Therefore, chromatin at the centro- and other non-histone tion of replacement variants, such as H3. 3 or CENP-A, meres contains one-half of the complement of CENP-A occurs outside S phasell 88. Thus, the histone variants nucleosomes after the completion of S phase and during H3. 3 and CENP-A have emerged as candidates for key subsequent G2 and M phases. To reconcile the deficit enzyme that catalyses the players of epigenetic information that can be transmitted in CENP-A molecules, current models predict that dur ing replication, either H3 1-containing nucleosomes specific Lys residues in histones are temporarily placed at centromeres, or, alternatively, nd other non-histone proteins. Inheritance of H3. 3. H3.3 is associated with transcrip- nucleosome gaps' are created that are filled later in the tionally active regions and is enriched in active histone cell cycle(FIG 4b) Heterochromatin protein 1 markssss9,90. Furthermore, nucleosomes that contain Recent studies suggest that CENP-A nucleosomes H3. 3 seem to be less stable than those that contain H3.1 are unusual and that these peculiarities might provide containing protein that binds (REF. 91). The extent to which this depends on the dif- a means of marking this region of the chromosome as ferential modification status of the nucleosomes, the unique. For example, in budding yeast, a specialized Cse4 presence of other variants, such as H2A.Z9, or inher- ( Saccharomyces cerevisiae CenH3)-containing nucleosome east(Swi6), mammals (HP1) ent differences in their structural properties remains has been proposed to exist in a form in which histon and Drosophila melanogaster to be established. Regardless, in vivo, these properties H2A and H2B are replaced by the non-histone protein suggest that H3. 3 nucleosomes are more dynamic or suppressor of chromosome missegregation 3( Scm3) Pericentric heterochromatin amenable to displacement during transcription. Given In D. melanogaster, ahemisome' that consists of one A heterochromatic region that replication leads to a concomitant deposition of molecule each of CenH3, H4, H2A and H2B has been H3. 1, the density of H33-containing nucleosomes described tor. Additional evidence suggests that, like H3.3 ontaining the centromere-spe. is reduced. As the mixing of H3. 1 and H3.3 in the nucleosomes, CENP-A nucleosomes are easier to dis- nd which is considered to be same nucleosome has not been observed.s,a semi- assemble in vitro than canonical nucleosomes.One conservative mechanism at the fork is unlikely for H3.3 might speculate that 'unusual CENP-A-containing nucleo- NATURE REVIEWS MOLECULAR CELL BIOLOGY 22009 Macmillan Publishers Limited All rights reserved

Histone deacetylase An enzyme that removes acetyl groups from histones. Lys methyltransferase An enzyme that catalyses the addition of a methyl group to specific Lys residues in histones and other non-histone proteins. Lys acetyltransferase An enzyme that catalyses the addition of an acetyl group to specific Lys residues in histones and other non-histone proteins. Heterochromatin protein 1 (HP1). A chromodomain￾containing protein that binds to methylated K9 on histone H3 and is associated with heterochromatin in fission yeast (swi6), mammals (HP1) and Drosophila melanogaster (HP1). Pericentric heterochromatin A heterochromatic region adjacent to chromatin containing the centromere-spe￾cific histone H3 variant CenH3, and which is considered to be typical constitutive heterochromatin. Particular features that are created at the time of DNA replication in a particular domain might also be exploited. NP95 has affinity for both hemimethylated DNA and histones34–36,84, and specifically interacts with peptides that are methylated at H3K9 in vitro85 by poten￾tially reading histone marks. In addition, NP95 was found in a complex with HDAcs and G9a31,32. Therefore, as well as binding to hemimethylated DNA, NP95 could interpret the histone environment, thereby creating a feedback mechanism that involves the mutual reinforce￾ment of histone and DNA methylation marks. In this situation, histone marks would influence the inheritance of DNA methylation. Further chromatin­binding pro￾teins or chromatin modifiers with dual affinity for both DNA methylation and a particular histone modification are likely to be identified. The examples above show how histone and DNA methylation in a repressive domain could be maintained at the replication fork. However, how active chromatin marks are propagated is less clear. Recently, transmission of an active state through nuclear transfer in Xenopus laevis has been reported, and it has been proposed that the replacement histone variant H3.3 is required for epi￾genetic memory86. To evaluate this hypothesis, it is nec￾essary to better understand the mechanisms that involve replication­independent histone exchange processes and the replacement of histone variants. inheritance of histone variants outside s phase Histone variants can mark a particular chromatin state: H3.3 is enriched at active regions, whereas the unique incorporation of the centromere­specific histone H3 variant cenH3 (ceNP­A in humans) specifies the site of centromere identity. Together with the replicative vari￾ants H3.1 and H3.2, the replacement variants H3.3 and ceNP­A constitute the major histone H3 isotypes that are known in mammals87. During S phase, H3.1 and H3.2 are exclusively incorporated, whereas the deposi￾tion of replacement variants, such as H3.3 or ceNP­A, occurs outside S phase11,88. Thus, the histone variants H3.3 and ceNP­A have emerged as candidates for key players of epigenetic information that can be transmitted in a replication­independent manner. Inheritance of H3.3. H3.3 is associated with transcrip￾tionally active regions and is enriched in active histone marks55,89,90. Furthermore, nucleosomes that contain H3.3 seem to be less stable than those that contain H3.1 (ReF. 91). The extent to which this depends on the dif￾ferential modification status of the nucleosomes55, the presence of other variants, such as H2A.Z92, or inher￾ent differences in their structural properties remains to be established. Regardless, in vivo, these properties suggest that H3.3 nucleosomes are more dynamic or amenable to displacement during transcription. Given that replication leads to a concomitant deposition of H3.1, the density of H3.3­containing nucleosomes is reduced. As the mixing of H3.1 and H3.3 in the same nucleosome has not been observed55,59, a semi￾conservative mechanism at the fork is unlikely for H3.3 inheritance (FIG. 3b). It is possible that the dilution of H3.3 and its marks by one­half after one cell cycle might not affect the transcriptional readout of a region. Thus, sustained active gene expression, combined with modifications on parental H3.3, might recruit factors that modify newly incorporated H3.1 with the appropriate marks, and H3.3 incorporation might be stimulated. consistent with this hypothesis, arrays of nucleosomes that contain both H3.3 and H3.1 nucleosomes have been observed, and analysis of histone modifications in this context show that, when adjacent to H3.3 nucleosomes, H3.1 nucleo￾somes accumulate active marks55. However, dilution of H3.3 over a number of generations might be reconciled by the replication­independent incorporation of H3.3 that is promoted by chaperones, such as Hir­related protein A (HIRA), following transcription59,93 (FIG. 4a). Inheritance of CENP‑A. The histone H3 variant ceNP­A marks the site of centromere identity94,95. The associa￾tion of ceNP­A with centromeres is extremely stable, as shown by quantitative fluorescence recovery after photo￾bleaching (FRAP) analysis, and it remains associated through cell division96. Although the exact mechanism of ceNP­A deposition at centromeres remains enigmatic, it is a replication­independent process, as is the deposition of H3.3 (ReF. 97). ceNP­A deposition was first proposed to occur in G2 phase, because ceNP­A assembly can take place in the presence of the DNA replication inhibitor aphidicolin, and CENPA mRNA and the ceNP­A pro￾tein peak in G2 phase97. Recent evidence in mammalian cells now suggests that the loading of new ceNP­A onto centromeres is restricted to a discrete cell cycle window in late telophase–early G1 phase98, but the mechanism and the specific chaperone that facilitate ceNP­A deposition remain to be deciphered. centromeric DNA is replicated during S phase, in which parental ceNP­A nucleosomes are distributed to daughter strands97,98. Therefore, chromatin at the centro￾meres contains one­half of the complement of ceNP­A nucleosomes after the completion of S phase and during subsequent G2 and m phases. To reconcile the deficit in ceNP­A molecules, current models predict that dur￾ing replication, either H3.1­containing nucleosomes are temporarily placed at centromeres, or, alternatively, nucleosome ‘gaps’ are created that are filled later in the cell cycle99 (FIG. 4b). Recent studies suggest that ceNP­A nucleosomes are unusual and that these peculiarities might provide a means of marking this region of the chromosome as unique. For example, in budding yeast, a specialized cse4 (Saccharomyces cerevisiaecenH3)­containing nucleosome has been proposed to exist in a form in which histones H2A and H2B are replaced by the non­histone protein suppressor of chromosome missegregation 3 (Scm3)100. In D. melanogaster, a ‘hemisome’ that consists of one molecule each of cenH3, H4, H2A and H2B has been described101. Additional evidence suggests that, like H3.3 nucleosomes, ceNP­A nucleosomes are easier to dis￾assemble in vitro than canonical nucleosomes102. One might speculate that ‘unusual’ ceNP­A­containing nucleo￾somes represent centromeric chromatin in an intermediate REVIEWS NATuRe ReVIeWS | Molecular cell Biology VOlume 10 | mARcH 2009 | 199 © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS In this respect, examining the distribution of particular histone variants at particular domains throughout the cell cycle might prove to be highly informative. Dilution of histone variants eplacement with H3.3 Challenges of heterochromatin maintenance during replicat Pericentric heterochromatin domains contribute to cor- rect chromosome segregation and must be maintained ●Q oughout the cell cycle. During mitosis and S phase, the particular molecular marks that characterize pericentric heterochromatin and its higher-order organization(BOX Modification of neighbouring H3. are challenged In different organisms, such as fission yeast and mice. diverse mechanisms have evolved that ensure OHZA-H2B ue身 More stable heterochromatin maintenance -New H3 HH4 T Active mark with H3.3-H4 with H3-H4 Fission yeast Fission yeast spends most of its lifetime in phase, during which pericentric repeats are organ zed into nucleosomes that are enriched in dimethylated H3K9(H3K9me2), to which the HPI homologue Swi6 is Centromeric chromati bound, Swi6 recruits the evolutionarily conserved ring shaped protein complex cohesin, which maintains sister cells enter 1 eeeeee.如油 plicatic 99自自自自 even before cytokinesis is completed o. The dilution of H3. 1 deposition repressive histone marks and further Swi6 delocaliza tion as a consequence of DNA replication are thought H3 eviction to allow access to the RNA polymerase II machiner and the bidirectional transcription of pericentromeric repeats occurs in this discrete cell cycle window of early S phase.I0(FIG5a) New H31H4 Indeed, a careful analysis of transcript levels during new CENP-A depositi the cell cycle reveals a correlation between the timings Figure 4 Inheritance of histone H3 variants outside of S phase. of replication and transcription, as the forward tran al Transcriptionally active domains are enriched in nucleosomes that contain histone scripts that have a transcription start site closer to the H3.3, which have more dynamic conformations and are enriched in active marks 58-1. replication origin accumulate first?. The transcripts Deposition of the histone variant H3. 1 during DNA replication results in the dilution of are processed into small interfering RNAs(siRNAs)that H3.3. Active marks on H3. 3 might recruit factors that facilitate the modification of accumulate transiently in S phase?. RNA interference neighbouring H3. 1 to ensure the inheritance of tive state in a dominant fashion (RNAi)-dependent and RNAi-independent mechanisms Loss of H3. 3 might be counterbalanced by the transcription-dependent incorporation then direct Lys methyltransferase(Clr4; also known function in chromatin assembly independently of DNA synthesis a 93.b incorporation Kmt D)and HDAC activity (Clr3 and Sir2), respectively. of the centromere-specific histone H3 variant CenH3(CENP-A in humans)at to re-establish heterochromatin characteristics follow centromeres is another DNA-synthesis-independent histone-deposition process" ing replication 09-ll. Whereas experimental evidence Replication of centromeric DNA in S phase dilutes CENP-A, resulting in three possible substantiates this model in yeast, whether RNAi is scenarios. First, parental CENP-A is equally distributed to daughter strands as a involved in heterochromatin maintenance in mam- dimer, possibly creating hemisomes. Second, parental CENP-A is distributed onto mals is unclear-ll6. Although pericentric repeats are daughter strands (as either tetramers or dimers)and H3. 1 is temporarily deposited at transcribed, not every component of the fission centromeres,resulting in asymmetric or random distribution. Third, parental CENP-A yeast RNAi machinery, such as RNA-dependent RNA is randomly distributed to daughter strands(as either tetramers or two dimers)and nucleosome 'gaps are created'run, Later in the cell cycle, during late telophase -early amplification of SIRNA production, has been identified specific deposition factors. Eviction of temporary H3. 1 from centromeres might precede in mammals Mice. As in fission yeast, mouse pericentric hetero- chromatin is enriched in HPl proteins, the binding of state that contains one-half of the amount of CENP-A, which is dependent on H3K9me3 as well as an uniden before it is fully replenished with new CENP-A mol- tified structural RNA component20, 12. Although ecules later in the cell cycle. Although the incorporation H3S10 phosphorylation occurs on entry into mitosis of replacement variants H3.3 or CENP-A is not directly in mammals 24123, some HPl is retained during mito dent on DNA replication, the distribution of paren- sis and, in contrast to fission yeast, it is enriched in histones at the fork could potentially pre-determine heterochromatin domains in Gl phase2412.Cell cycle how and when H3. 1 can be replaced at later stages. regulation of the transcription of pericentric repeats 200 MAR 22009 Macmillan Publishers Limited All rights reserved

Nature Reviews | Molecular Cell Biology Transcription-coupled Dilution of histone v replacement with H3.3 ariants during replication HIRA 1 2 ‘Hemisome’ Temporary H3.1 deposition 3 Late telophase–early G1; new CENP-A deposition ‘Gaps’ Centromeric chromatin H3.3–H4 H2A–H2B ‘Unstable’ nucleosome with H3.3–H4 More stable nucleosome New H3. with H3.1–H4 1–H4 Active mark a b CENP-A–H4 New H3.1–H4 S phase; CENP-A dilution Modification of neighbouring H3.1 X H3.1 eviction or state that contains one­half of the amount of ceNP­A, before it is fully replenished with new ceNP­A mol￾ecules later in the cell cycle. Although the incorporation of replacement variants H3.3 or ceNP­A is not directly dependent on DNA replication, the distribution of paren￾tal histones at the fork could potentially pre­determine how and when H3.1 can be replaced at later stages. In this respect, examining the distribution of particular histone variants at particular domains throughout the cell cycle might prove to be highly informative. Challenges of heterochromatin maintenance Pericentric heterochromatin domains contribute to cor￾rect chromosome segregation and must be maintained throughout the cell cycle. During mitosis and S phase, the particular molecular marks that characterize pericentric heterochromatin and its higher­order organization (BOX 2) are challenged. In different organisms, such as fission yeast and mice, diverse mechanisms have evolved that ensure heterochromatin maintenance. Fission yeast. Fission yeast spends most of its lifetime in G2 phase, during which pericentric repeats are organ￾ized into nucleosomes that are enriched in dimethylated H3K9 (H3K9me2), to which the HP1 homologue Swi6 is bound. Swi6 recruits the evolutionarily conserved ring￾shaped protein complex cohesin, which maintains sister chromatid cohesion103,104. As cells enter mitosis, histone H3 becomes phosphorylated on residue S10, which results in reduced Swi6 binding and facilitates chromo￾some segregation105–107. centromeres undergo replication even before cytokinesis is completed108. The dilution of repressive histone marks and further Swi6 delocaliza￾tion as a consequence of DNA replication are thought to allow access to the RNA polymerase II machinery, and the bidirectional transcription of pericentromeric repeats occurs in this discrete cell cycle window of early S phase106,107 (FIG. 5a). Indeed, a careful analysis of transcript levels during the cell cycle reveals a correlation between the timings of replication and transcription, as the forward tran￾scripts that have a transcription start site closer to the replication origin accumulate first107. The transcripts are processed into small interfering RNAs (siRNAs) that accumulate transiently in S phase107. RNA interference (RNAi)­dependent and RNAi­independent mechanisms then direct lys methyltransferase (clr4; also known as Kmt1) and HDAc activity (clr3 and Sir2), respectively, to re­establish heterochromatin characteristics follow￾ing replication109–113. Whereas experimental evidence substantiates this model in yeast, whether RNAi is involved in heterochromatin maintenance in mam￾mals is unclear114–116. Although pericentric repeats are transcribed117,118, not every component of the fission yeast RNAi machinery, such as RNA­dependent RNA polymerase, which serves in the post­transcriptional amplification of siRNA production, has been identified in mammals119. Mice. As in fission yeast, mouse pericentric hetero￾chromatin is enriched in HP1 proteins, the binding of which is dependent on H3K9me3 as well as an uniden￾tified structural RNA component 120,121. Although H3S10 phosphorylation occurs on entry into mitosis in mammals122,123, some HP1 is retained during mito￾sis and, in contrast to fission yeast, it is enriched in heterochromatin domains in G1 phase124,125. cell cycle regulation of the transcription of pericentric repeats Figure 4 | inheritance of histone H3 variants outside of S phase. a | Transcriptionally active domains are enriched in nucleosomes that contain histone H3.3, which have more dynamic conformations and are enriched in active marks55,89–91. Deposition of the histone variant H3.1 during DNA replication results in the dilution of H3.3. Active marks on H3.3 might recruit factors that facilitate the modification of neighbouring H3.1 to ensure the inheritance of an active state in a dominant fashion. Loss of H3.3 might be counterbalanced by the transcription-dependent incorporation of H3.3 promoted by histone chaperones, such as Hir-related protein A (HIRA), that function in chromatin assembly independently of DNA synthesis59,89,93. b | Incorporation of the centromere-specific histone H3 variant CenH3 (CENP-A in humans) at centromeres is another DNA-synthesis-independent histone-deposition process97. Replication of centromeric DNA in S phase dilutes CENP-A, resulting in three possible scenarios. First, parental CENP-A is equally distributed to daughter strands as a dimer, possibly creating hemisomes. Second, parental CENP-A is distributed onto daughter strands (as either tetramers or dimers) and H3.1 is temporarily deposited at centromeres, resulting in asymmetric or random distribution. Third, parental CENP-A is randomly distributed to daughter strands (as either tetramers or two dimers) and nucleosome ‘gaps’ are created99,101. Later in the cell cycle, during late telophase–early G1 phase, newly synthesized CENP-A is deposited at centromeres98, possibly by specific deposition factors. Eviction of temporary H3.1 from centromeres might precede the deposition of new CENP-A. REVIEWS 200 | mARcH 2009 | VOlume 10 www.nature.com/reviews/molcellbio © 2009 Macmillan Publishers Limited. All rights reserved

REVIEWS a Fission yeast b mice mere Pericentromen M Phospho- 888g. ee RNAI 9 DNA methylation -PEricentric O Histone methylation e Histone phosphorylation P New mark Figure 5 Maintaining pericentric heterochromatin in fission yeast and mouse. Cell cycle profiles and the timing of pericentric heterochromatin replication differ between fission yeast and mammals. a In G2 phase in fission yeast, heterochromatin protein 1(HP1)homologue Swi6(REF. 69). On entry into mitosis, phosphorylation of H3S10 leads reduced Swi6 association. This is termed the phospho-methyl switch0s-107 Centromeric repeats are transcribed after centromere replication in early S phase and after dilution of histone marks 04lo? Transcripts are processed by the rna interference(RNAi ry into small interfering RNAs(siRNAs). The RNAi machinery recruits the histone methyltransferase CIr4(the Suv 39 homologue)tom Deacetylation by Clr3 and H3K9 dimethylation by CIr4 lead to the restoration of Swi6 binding and to silent heterochromatin maintenance. b Pericentric heterochromatin in mice contains methylated DNA and H3K9me3, which is bound by HP1 (REF. 70). The extent to which HP1 is disrupted by the ho-me vitch and how HPl is restored in Gl phase is unclear 27-125 Centromeric transcripts accumulate in mitosis and in Gl-early S phase126 however, a direct role for RNA in heterochromatin maintenance in mice is lacking Maintenance of pericentric heterochromatin occurs through the concerted action of DNA and histone modifiers an histone chaperones: DNA methylation is maintained by DNA methyltransferase 1(DNMT1), which, together with proliferating cell nuclear antigen(PCNA), recruits histone deacetylase activity Bs1B2 Chromatin assembly factor 1 (CAF1: also known as CHAF1)ensures histone H3. 1 deposition and HP1 inheritance by the transfer of parental HP1to daughter strands 2A1s0, where it is maintained by a self-perpetuating loop that involves SUV39H1 (also known as KMT1A67-/0. I8L H3 1 can be monomethylated at residue k9 before deposition, serving as a substrate for further modification in chromatin. HDAC, histone deacetyla was also documented in mice2. Two RNA species By contrast, the transmission and silencing of hetero- were identified: a short species that accumulates spe- chromatin in mice could be ensured by mutual rein cifically in mitosis and another species of variable size forcement between the inheritance of DNA and histone Small interfering RNA that accumulates in Gl phase and peaks at Gl-S phase. modifications at the replication fork (FIG 5b). The main Whether the short pericentric transcripts have a role in tenance of heterochromatin in mammals requires dNA (w 22-nt long) that is processed HPI dynamics during mitosis is unknown. However, methylation, histone deacetylation, H3K9 trimethylation from longer double-stranded the transcription of the longer species was found to and the transmission of HPI proteins to the daughter RNA by the RNa interfere ence cease before replication of heterochromatin domains, strands. As discussed above, DNMTI is enriched at machinery. Such non-coding which renders a direct role for pericentric transcripts pericentric heterochromatin in mid-S p the silencing complexes in in post-replicative maturation of heterochromatin in both PCNA and DNMTI recruit HDAC activity. CAFI which they resid is present in two mutually exclusive complexes, either NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 10 I MARCH 20091201 22009 Macmillan Publishers Limited All rights reserved

Nature Reviews | Molecular Cell Biology Swi6 P P Phospho–methyl switch Phospho– methyl switch G2 M G1 G2 M G1 Early S Clr4 siRNAs RNAi machinery New mark DNA methylation Histone methylation mark Histone phosphorylation mark HP1 ? Mid S HDAC DNMT1 HDAC CAF1 CAF1 CAF1 SUV39H ? ? PCNA Pericentric transcripts G2 G2 S S M M G1 G1 a Fission yeast b Mice Pericentromere replication Pericentromere replication P P P P Clr3 P Small interfering RNA A short, non-coding RNA (~22-nt long) that is processed from longer double-stranded RNA by the RNA interference machinery. such non-coding RNAs confer target specificity to the silencing complexes in which they reside. was also documented in mice126. Two RNA species were identified: a short species that accumulates spe￾cifically in mitosis and another species of variable size that accumulates in G1 phase and peaks at G1–S phase. Whether the short pericentric transcripts have a role in HP1 dynamics during mitosis is unknown. However, the transcription of the longer species was found to cease before replication of heterochromatin domains, which renders a direct role for pericentric transcripts in post­replicative maturation of heterochromatin in mouse unlikely126. By contrast, the transmission and silencing of hetero￾chromatin in mice could be ensured by mutual rein￾forcement between the inheritance of DNA and histone modifications at the replication fork (FIG. 5b). The main￾tenance of heterochromatin in mammals requires DNA methylation, histone deacetylation, H3K9 trimethylation and the transmission of HP1 proteins to the daughter strands. As discussed above, DNmT1 is enriched at pericentric heterochromatin in mid­S phase127 , and both PcNA and DNmT1 recruit HDAc activity. cAF1 is present in two mutually exclusive complexes, either Figure 5 | Maintaining pericentric heterochromatin in fission yeast and mouse. Cell cycle profiles and the timing of pericentric heterochromatin replication differ between fission yeast and mammals. a | In G2 phase in fission yeast, pericentric hetrochromatin is enriched in dimethylated histone H3K9 (H3K9me2), which provides a binding site for the heterochromatin protein 1 (HP1) homologue Swi6 (ReF. 69). On entry into mitosis, phosphorylation of H3S10 leads to reduced Swi6 association. This is termed the phospho–methyl switch105–107. Centromeric repeats are transcribed after centromere replication in early S phase and after dilution of histone marks106,107. Transcripts are processed by the RNA interference (RNAi) machinery into small interfering RNAs (siRNAs). The RNAi machinery recruits the histone methyltransferase Clr4 (the Suv39 homologue)109. Deacetylation by Clr3 and H3K9 dimethylation by Clr4 lead to the restoration of Swi6 binding and to silent heterochromatin maintenance113. b | Pericentric heterochromatin in mice contains methylated DNA and H3K9me3, which is bound by HP1 (ReF. 70). The extent to which HP1 is disrupted by the phospho–methyl switch and how HP1 is restored in G1 phase is unclear122–125. Centromeric transcripts accumulate in mitosis and in G1–early S phase126; however, a direct role for RNA in heterochromatin maintenance in mice is lacking. Maintenance of pericentric heterochromatin occurs through the concerted action of DNA and histone modifiers and histone chaperones: DNA methylation is maintained by DNA methyltransferase 1 (DNMT1), which, together with proliferating cell nuclear antigen (PCNA), recruits histone deacetylase activity78,81,82. Chromatin assembly factor 1 (CAF1; also known as CHAF1) ensures histone H3.1 deposition and HP1 inheritance by the transfer of parental HP1 to daughter strands128,130, where it is maintained by a self-perpetuating loop that involves SUV39H1 (also known as KMT1A)67–70,181. H3.1 can be monomethylated at residue K9 before deposition, serving as a substrate for further modification in chromatin55. HDAC, histone deacetylase. REVIEWS NATuRe ReVIeWS | Molecular cell Biology VOlume 10 | mARcH 2009 | 201 © 2009 Macmillan Publishers Limited. All rights reserved