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 reservedNature 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