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 reservedNature 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