《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