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Chromatin is organized into distinct functional domains. During mitotic cell division, both genetic information encoded in DNA sequence and epigenetic information embedded in chromatin structure must be faithfully duplicated. The inheritance of epigenetic states is critical in maintaining the genome integrity and gene expression state. In this review, we will discuss recent progress on how proteins known to be involved in DNA replication and DNA replication-coupled nucleosome assembly impact on the inheritance and maintenance of heterochromatin, a tightly compact chromatin structure that silences gene transcription. As heterochromatin is important in regulating gene expression and maintaining genome stability, understanding how heterochromatin states are inherited during S phase of the cell cycle is of fundamental importance.
In eukaryotic cells, DNA and its associated proteins form chromatin, which undergoes extensive compaction to fit within the cell nucleus. The basic repeating unit of chromatin is the nucleosome, which consists of two copies each of four histones (H3, H4, H2A, and H2B) wrapped by 147 bp of DNA . Nucleosomes are further packaged into higher-order chromatin structures. The organization of DNA into chromatin functions to protect genetic information as well as to provide a means by which the cell can regulate gene activity. Chromatin is regulated in multiple ways. First, histones are modified post-translationally, including acetylation, phosphorylation, methylation, ubiquitination, and other histone modifications [2,3]. Second, in addition to canonical histones, chromatin is demarcated by histone variants, a group of proteins that adopt similar folds as canonical histones and perform unique functions that cannot be substituted by canonical histones . Third, the DNA sequence itself can be modified by DNA cytosine methylation and hydromethylation [5,6]. Finally, chromatin is regulated/modified by non-coding RNAs . Historically, chromatin is classified into two major types, heterochromatin and euchromatin, based on the cytological staining. Histone modifications, histone variants, DNA modifications, and non-coding RNAs function alone and/or in concert to separate chromatin into distinct functional domains [3,8,9].
In addition to the regulation of chromatin organization, histone and DNA modifications, non-coding RNAs and histone variants regulate gene activity, possibly via a DNA-sequence-independent manner, which in turn generates epigenetic information that governs cell identity and gene expression states. Finally, chromatin structures also play regulatory roles in other cellular processes such as DNA replication, DNA repair, and chromosome segregation. Thus, chromatin and its constituents play fundamental roles in almost every aspect of cell biology.
During mitotic cell division, not only must the DNA sequence be faithfully replicated and transmitted to daughter cells, but the chromatin structure must also be propagated for transmission of epigenetic information . How epigenetically determined chromatin domains, which are composed of distinct histone modifications, non-coding RNAs, histone variants, and/or DNA modifications, are inherited during S phase of the cell cycle remains largely unknown. In this review, we discuss proteins known to be involved in DNA replication and DNA replication-coupled (RC) nucleosome assembly and their roles in the inheritance and maintenance of heterochromatin. As heterochromatin is important in regulating gene expression and maintaining genome stability, understanding how heterochromatin states are inherited during S phase of the cell cycle is of fundamental importance.
In eukaryotic cells, DNA replication is initiated by the concerted actions of proteins involved in origin recognition, DNA unwinding, and cell cycle control (Fig. 1). Initiation of DNA replication begins with the binding of the origin recognition complex (ORC)  to DNA replication origins. The ORC complex is a six-subunit complex (Orc1–Orc6) conserved from yeast to humans and is absolutely required for replication initiation [12,13]. Following the binding of ORC to replication origins, Cdc6 and Cdt1 coordinate to load the replicative helicase, MCM (mini chromosome maintenance) protein complex, to origins. The MCM complex is composed of six related proteins (Mcm2, Mcm3, Mcm4, Mcm5, Mcm6, and Mcm7) and is loaded as head-to-head double hexamers around double-stranded DNA . With the help of two kinases, DDK and CDK, the MCM helicase activity is activated, and by association with Cdc45 and GINS, a large and active helicase complex called CMG (Cdc45/MCM2-7/GINS) is formed. CMG unwinds duplex DNA into single-stranded DNA, which is then coated by the single-strand DNA-binding protein (RPA) . Mcm10 is required for stabilization of the DNA polymerase α (Pol α)-primase complex that synthesizes a short RNA–DNA segment as the primer for DNA synthesis. Replication factor C (RFC), a protein complex consisting of five subunits, then loads proliferating cell nuclear antigen (PCNA) onto the primer-template junction . PCNA serves as the processivity factor for DNA polymerase, Pol δ or Pol , polymerases replicating the lagging and leading strand, respectively [17,18], and as a platform for the recruitment of a variety of proteins involved in DNA replication . Thus, DNA replication is achieved through the coordinated action of multiple functional machines.
Given its responsibility in replicating DNA at all chromatin domains, it is not unprecedented that the DNA replication machinery functions in the inheritance of chromatin domains. Indeed, as discussed below, proteins involved in DNA replication play an important role in the formation and maintenance of heterochromatin, and some of these roles are independent from the DNA replication function. Mechanistically, heterochromatin formation is better understood in budding and fission yeast, so we will briefly outline key components and properties of heterochromatin in these two yeast species and then discuss the roles of several DNA replication proteins in heterochromatin silencing in higher eucaryotic cells.
In budding yeast, Saccharomyces cerevisiae, heterochromatin-like structures termed as silent chromatin are found at telomeres, the silent mating-type loci, HMR and HML, and the rDNA locus. The silent chromatin structure can influence the expression of genes located within or surrounding these loci. Formation of silent chromatin at these three loci serves different purposes and requires different protein components [20,21]. Transcriptional silencing at the HM loci requires four silent information regulator proteins (Sir1p, Sir2p, Sir3p, and Sir4p). Silencing at the HM loci initiates through the recruitment of Sir proteins to the E silencer, a DNA sequence element that contains a DNA-binding consensus sequence for the transcriptional factors Abf1, Rap1, and ORC. Once recruited to the silencer, Sir2p, a nicotinamide adenine dinucleotide-dependent histone deacetylase [22,23] specific for acetyl-lysines 9 and 14 of H3 and 16 of H4 [22–25], deacetylates histones of nearby nucleosomes to establish a binding site for Sir3 and Sir4, which exhibit higher binding affinity for hypoacetylated histones [26,27]. Repeating the sequential recruitment of Sir proteins and histone deacetylation allows for the spreading of Sir proteins to the whole silent chromatin domain [20,28–30]. The spreading of Sir proteins is terminated by boundary elements at the HM loci [31–33]. While silencing at telomeres follows a similar mechanism of initiation and spreading as the HM loci, spreading is terminated by Sas2p, a histone acetyltransferase targeting histone H4 lysine 16 (H4K16), which creates a histone acetylation gradient, restricting Sir proteins to the telomeric heterochromatin [31,32]. The budding yeast has served as a simple model for studying how heterochromatin is formed and maintained and has offered much insight into the heterochromatin structure.
In Schizosaccharomyces pombe and higher eukaryotic cells, heterochromatin is marked by histone H3 lysine 9 methylation (H3K9me). Histone H3K9 is methylated by Clr4/Suv39h [34,35] in fission yeast and by histone methyltransferase Suv39h1/h2 at pericentromeric regions in mammalian cells . H3K9me2/3 provides a binding site for the heterochromatin protein Swi6, the sequence and functional homolog of HP1 in higher eukaryotic cells [37–41]. HP1 was first identified as a component of a tightly bound non-histone chromosomal protein fraction from Drosophila embryo nuclei  and later was determined to be identical to Su(var) 2–5, a dominant suppressor of position effect variegation (PEV) [43,44]. Swi6 and HP1 contain an N-terminal chromodomain, a flexible hinge region and a C-terminal chromoshadow domain (CSD). The chromodomain of Swi6 and HP1 functions as a binding module for H3K9me3 [45–47]. Mammalian cells contain three isoforms of HP1 proteins, HP1α, HP1β, and HP1γ, which all localize to pericentrimeric heterochromatin to different degrees . Binding of HP1 family proteins to pericentrimeric heterochromatin thus results in the repression of gene expression within these regions .
In addition to DNA sequence-specific binding proteins, RNA interference (RNAi) also plays an important role in establishing and maintaining heterochromatin at centromeres in S. pombe [50–52]. Each S. pombe centromere contains a unique kinetochore-associated DNA sequence termed as the central core region (cnt), which is defined by the histone variant CENP-A. The cnt region is flanked by two types of large and repetitive sequences organized into innermost (imr) and outermost (otr) repeats. The otr regions consist of dg and dh repeats . Along with the enrichment of Swi6 on the dg/dh repeats within the pericentromeric regions, RNA polymerase II transcribes the dg/dh repeats, which are degraded by the small interfering RNA (siRNA)programmed RITS (RNA-induced initiator of transcriptional silencing) complex [35,54,55]. Moreover, the RITS complex recruits the RNA-dependent RNA polymerase and dicer (Dcr1) ribonuclease, which generates siRNAs. This process is called post-transcriptional silencing in cis . RITS contains Argonaute (Ago1), the chromodomain protein Chp1, and the glycine-tryptophan (GW) domain protein Tas3, which directly recruits the CLRC complex containing the Clr4/Suv39h methyltransferase. In turn, methylation of H3K9 promotes the chromatin binding of RITS [51,56–58].
Formation of silent chromatin at the HM loci in budding yeast requires passage through S phase as well as M phase of the cell cycle . Studies indicate that DNA replication itself is not important, as silent chromatin can still form on DNA that is excised from chromosomes and does not contain a replication origin [59,60]. While DNA replication per se may not be essential for formation of silent chromatin in budding yeast, several proteins involved in DNA replication, as detailed below, are important for heterochromatin silencing in budding yeast and other organisms.
The N-terminus of Orc1, which shares sequence homology with Sir3 and contains the bromo-adjacent homology (BAH) domain, binds Sir1, and recruits Sir1 to the E silencer [61,62]. In addition, the BAH domain is dispensable for replication, indicating that Orc1 has a role in transcriptional silencing that is independent of its role in DNA replication . In addition to Orc1, mutations in other ORC subunits, such as Orc2, affect transcriptional silencing [64–66]. Compromised ORC function is suppressed by inactivation of Sir2 . Because cells harboring Orc2 mutations also exhibit defects in DNA replication and mitosis [64–66], how different ORC mutations affect transcriptional silencing in budding yeast is not clear.
The role of ORC in heterochromatin formation and silencing is likely to be conserved. In S. pombe, ORC interacts with Swi6 and plays an important role in centromere and mating-type gene silencing . In Drosophila, DmOrc2 interacts with HP1 [69–71]. Similarly, human Orc2 (hOrc2) binds to HP1 α and HP1 β in G1 and early S phase  and co-localizes with HP1 at pericentric heterochromatin. Depletion of Orc2 affects the localization of HP1 to heterochromatin, but not the levels of H3K9me3 , suggesting that the heterochromatin association of HP1 depends on both H3K9me3 and ORC. Supporting this idea, a genome-wide screen for genes involved in transcriptional silencing on the inactivated X-chromosomes (Xi) in female mouse embryonic fibroblast cells revealed that Orc2 is required for the efficient silencing of Xi genes and for the stable association of HP1 α on Xi . Thus, Orc2 and possibly other subunits of the ORC complex are involved in the formation and/or maintenance of constitutive heterochromatin (such as pericentric heterochromatin) and facultative heterochromatin (such as Xi).
In addition to ORC, the leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1/ORCA), a protein that interacts with ORC, is also localized to heterochromatic foci . Interestingly, using quantitative mass spectrometry in combination with genome-wide profiling of epigenetic marks, both ORC and Lrwd1 were found to preferentially bind nucleosomes containing heterochromatin marks (H3K9me3 or H3K27me3), raising the possibility that ORC and Lrwd1 are recruited to heterochromatin foci through interactions with repressive histone marks [76,77]. However, it is not known how the ORC-Lrwd1 complex recognizes heterochromatin marks or why it is important for ORC and Lrwd1 to associate with heterochromatin and stabilize HP1 on heterochromatin. Additional studies are needed to address these questions.
In addition to ORC, roles for DNA polymerases have been demonstrated in heterochromatin silencing in both S. cerevisiae and S. pombe. Through surveying the effect of 41 mutations in genes involved in DNA replication, cell cycle and DNA repair on silencing at the HMR locus, mutations at three subunits of DNA Pol ε were identified, suggesting a role for Pol ε in HMR silencing ; however, how DNA polymerase ε is involved in transcriptional silencing is not well studied. Recently, it has been shown that Cdc20, the catalytic subunit of DNA Pol ε in S. pombe, co-purifies with Dos2, Rik1, and Mms19 . Dos2 and Rik1 are involved in promoting Clr4-mediated methylation of H3 lysine 9. Compromising the function of Cdc20 results in defects in centromere transcriptional silencing with reduced levels of H3K9me3 at centromeric heterochromatin [80–82]. The Dos2, Rik1, Mms19, and Cdc20 complex regulates the transcription of siRNA at heterochromatin foci during S phase, thus connecting DNA replication to the maintenance of heterochromatin silencing in S. pombe .
In addition to Pol ε, mutations in the catalytic subunit of DNA Pol α in S. pombe result in reduced Swi6 association at all three heterochromatin loci, mating-type region, centromere, and telomeres. Moreover, Pol α physically interacts with Swi6 in vitro. These results suggest that Pol α has a role in heterochromatin assembly and/or maintenance. Because Pol α's primary role is in lagging strand DNA synthesis, it is possible that Pol α and Pol ε impact on heterochromatin formation and maintenance during lagging and leading strand DNA synthesis, respectively. Future studies are needed to determine how these DNA polymerases function in the maintenance and inheritance of heterochromatin.
PCNA is also involved in heterochromatin silencing and/or chromatin dynamics. PCNA, encoded by POL30 in S. cerevisiae, is the processivity factor that tethers DNA polymerase δ and ε to the replication fork and interacts with many proteins involved in DNA replication [83,84]. Mutations in PCNA result in defects in transcriptional silencing at telomeres and the silent mating-type HMR locus . In Drosophila, mutation of the PCNA ortholog mus209 suppresses PEV . Furthermore, different pol30 alleles affect transcriptional silencing distinctly, suggesting that PCNA regulates heterochromatin silencing through multiple mechanisms. Consistent with this idea, PCNA interacts with several proteins involved in chromatin dynamics. For instance, studies from our laboratory show that PCNA directly interacts with the histone/protein acetyltransferase elongator complex both in vivo and in vitro . In addition, pol30 mutants genetically interact with histone modifications linked to ASF1 and CAF-1-dependent pathways, including SASI- and Rtt109p-dependent acetylation events at H4K16, H3K9, and H3K56 [88,89]. In mammalian cells, PCNA interacts with several chromatin modification enzymes. For example, PCNA interacts with DNMT1, a DNA methyltransferase that methylates hemi-methylated DNA [90,91]. PCNA also binds SET8/Pr-Set7, an H4K20-specific methyltransferase that monomethylates H4K20 (H4K20me) during the S phase . H4K20me is a critical substrate for trimethylation of H4K20 (H4K20me3), a mark associated with constitutive pericentromeric heterochromatin formation [93,94]. Given the central role of PCNA at the replication fork , PCNA may orchestrate proteins involved in DNA replication, nucleosome assembly (see below), histone modification, and DNA methylation to ensure faithful transmission of both genetic and epigenetic information.
In addition to ORC, PCNA, and DNA polymerases, mutations in other proteins involved in DNA replication also affect heterochromatin silencing. For instance, in budding yeast, mutations in CDC45 and RF-C (CDC44) restore silencing at a mutant HMR silencer allele . Moreover, mutation of CDC7, encoding a protein kinase required for the initiation of DNA replication, restores silencing at the alpha mating type ; Furthermore, mutations in MCM5 result in elevated expression of sub-telomeric and Ty retrotransposon-proximal genes , and mutations in MCM10 significantly reduce silencing at both telomeres and HM loci . Taken together, these genetic studies further support the hypothesis that DNA replication factors function in heterochromatin silencing. In addition to these studies, several protein factors have been found to bind heterochromatin proteins. For instance, Mcm10 physically interacts with Sir2 in budding yeast [97–99], and Drosophila Mcm10 interacts with HP1 . In S. pombe, Cdc18 (ScCdc6) interacts with Swi6 (HP1) . Biochemical mapping and mutational analysis demonstrate that the N-terminus of Cdc18 interacts with the chromoshadow domain of Swi6. Interestingly, mutations in the binding domain of Cdc18 and Swi6 alter the distribution of Swi6 at specific centromere regions as well as the timing of centromere replication . Using a yeast two-hybrid assay in combination with chromatin immunoprecipitation, HP1 has been found to interact with Cdc6, Cdc45, and RPA in mouse cells . Thus, many protein factors involved in DNA replication have been documented to affect heterochromatin silencing from yeast to human cells, some of which directly interact with proteins required for the establishment and maintenance of heterochromatin.
Nucleosomes pose a ‘barrier’ for the DNA replication machinery; therefore, nucleosomes ahead of DNA replication fork are temporarily disassembled to facilitate the progression of replication machinery. Following DNA replication, replicated DNA is assembled into nucleosomes with incorporation of both parental and new histones. This nucleosome disassembly/reassembly process coupled to ongoing DNA replication is called DNA RC nucleosome assembly (Fig. 2) [103,104]. At the molecular level, nucleosomes are assembled in a stepwise fashion with deposition of (H3-H4)2 tetramers first followed by rapid deposition of two H2A-H2B dimers [105,106]. Moreover, parental (H3-H4)2 tetramers are transferred as a unit onto replicating DNA to form nucleosomes . It remains mechanistically unclear as to how parental (H3-H4)2 tetramers are transferred. On the other hand, several histone chaperones have been described that facilitate the deposition of newly synthesized (H3-H4)2 tetramers onto DNA to promote what is termed as de novo nucleosome assembly. Therefore, we will focus our discussion on factors involved in de novo nucleosome assembly and their function in heterochromatin silencing in diverse organisms.
One of the central histone chaperones in RC nucleosome assembly is chromatin assembly factor 1 (CAF-1), a three-subunit protein complex conserved among eukaryotes . CAF-1 binds H3-H4 and preferentially assembles replicating DNA into nucleosomes in a reaction dependent upon the CAF-1–PCNA interaction. Various studies indicate that mutations in CAF-1 result in partial loss of transcriptional silencing at silent chromatin or a silenced transgene from yeast to mammalian cells [109–113]. In budding yeast, deletion of CAC1, CAC2, or CAC3 reduces gene silencing at telomeres [114,115]. In S. pombe, depletion of spCAF-1 causes defects in silencing at the centromere and mating-type locus . In Drosophila, a reduction of Drosophila p180, the largest subunit of CAF-1, suppress gene silencing at heterochromatin [113,117] and results in a decrease in H3K9 methylation at pericentric heterochromatin and compromised recruitment of HP1 to the chromocenter of the polytene chromosomes . In mice, loss of CAF-1 p150 leads to severe alterations in constitutive heterochromatin structure and developmental arrest at the 16-cell stage , indicated that CAF-1 p150 is specifically required for heterochromatin organization in pluripotent embryonic cells. CAF-1 likely participates in heterochromatin formation and inheritance via two non-overlapping mechanisms. First, CAF-1's role in nucleosome assembly is important for heterochromatin silencing. In this regard, it is known that loss of nucleosome density prevents the spreading of heterochromatin. We have shown that yeast cells lacking CAF-1 and Rtt106, another histone H3-H4 chaperone, exhibit defects in Sir protein spreading during re-establishment of silent chromatin in budding yeast . Second, CAF-1 interacts with proteins involved in heterochromatin silencing and/or maintenance and may be required for recruitment of these proteins during DNA replication for reassembly of heterochromatin. Supporting this idea, it has been shown that CAF-1 interacts with HP1 in mammalian cells. Interestingly, ectopic expression of HP1 in a euchromatin location leads to enrichment of Drosophila CAF-1 p180 at this site, suggested a mutual recruitment of HP1 and CAF-1 p180 to chromatin . Finally, CAF-1 is proposed to recruit the SETDB1 methyltransferase complex for methylation of new histones at H3K9 through formation of the CAF-1-MBD-SETDB1 complex  and the HP1a-CAF-1-SETDB1 further promotes trimethylation of H3K9me1 by Suv39H1/H2 in pericentric regions , indicating a highly coordinated mechanism to ensure the propagation of epigenetic marks of pericentric heterochromatin during DNA replication. Taken together, it is possible that CAF-1 couples nucleosome assembly with the recruitment of HP1/Swi6 and histone methyltransferases for inheritance of heterochromatin during de novo nucleosome assembly.
Another well-documented histone chaperone in RC nucleosome assembly is anti-silencing factor 1 (Asf1). Asf1 was first identified as a disruptor of gene silencing when overexpressed in budding yeast [121,122]. Deletion of ASF1 displays only minor defects in transcriptional silencing [121,122]. When combined with mutations in Cac1, the large subunit of CAF-1, asf1 dramatically affects gene silencing at silent chromatin , suggesting that CAF-1 and Asf1 function in different pathways to regulate heterochromatic gene silencing. More recently, an elegant study in fission yeast demonstrated that a histone chaperone complex containing Asf1 and histone regulatory homolog A (HIRA) spreads the heterochromatin domains via an interaction with Swi6/HP1 at heterochromatic loci . HIRA is known to function in replication-independent nucleosome assembly  and is also involved in silencing in budding yeast [126–129]. Interestingly, Asf1 co-purifies with the Clr6 HDAC complex and facilitates histone deacetylation on chromatin. Moreover, micrococcal nuclease (MNase) digestion in combination with microarray analysis (MNase-Chip) revealed that deletion of ASF1 results in reduced nucleosome occupancy at heterochromatic loci in S. pombe, consistent with the idea that Asf1 likely functions in silencing by regulating nucleosome occupancy at heterochromatin loci.
Rtt106, another H3-H4 chaperone, also functions in heterochromatin silencing in budding yeast [118,130]. Rtt106 was identified in a screen for enhancers of heterochromatic silencing defects of a PCNA mutant . Deletion of RTT106 synergistically enhances the silencing defects of cac1, but not asf1 mutant cells, suggesting that Rtt106 and CAF-1 function in parallel pathways in heterochromatin silencing. In addition, while deletion of CAC1, the largest subunit of CAF-1, has only minor effects on Sir protein localization to heterochromatin loci [131,132], deletion of both CAC1 and RTT106 results in a significant reduction of Sir4, Sir2, and Sir3 at telomeric heterochromatin . Moreover, Rtt106 interacts with Sir4 both in vivo and in vitro . Structural and functional analyses of Rtt106 revealed that the ability of Rtt106 to bind H3-H4 is required for Rtt106's role in gene silencing (Su D et al., unpublished results; ). Together, these results suggest that Rtt106 likely functions in heterochromatin silencing through its ability to assemble nucleosomes as well as its ability to interact with Sir proteins. While the sequence homolog of Rtt106 has not been identified in mammalian cells, Daxx, a histone chaperone that deposits the histone H3 variant H3.3 onto chromatin in a replication-independent manner, contains a region similar to Rtt106. Moreover, Daxx is required for localization of H3.3 at telomeric heterochromatin in mouse embryonic fibroblast cells [134,135]. It is unknown to what extent Daxx is also involved in heterochromatin silencing in mammalian cells.
FACT is another histone chaperone that may function in RC nucleosome assembly. FACT (facilitates chromatin transcription) was first identified through its ability to enhance transcription of chromatin templates by RNA polymerase II and consists of two subunits, Spt16 and SSRP1/Pob3 . FACT binds both H2A-H2B dimers and H3-H4 tetramers in vitro [137,138]. In budding yeast, Spt16 interacts with something about silencing (Sas3), the catalytic subunit of the NuA3 HAT complex, in vitro and in vivo. Mutations in SAS3 restore silencing to a derepressed HMR locus, but enhance silencing defects at the HML silent mating locus . In fission yeast, FACT co-purifies with Swi6/HP1 , and deletion of Pob3 results in reduced silencing at centromeric repeats and the mating-type locus with compromised association of Swi6 to heterochromatin . More recently, FACT was found to interact with Drosophila HP1 by association of the central region of dSSRP1 (homolog of Pob3) and the CSD of HP1 . These results demonstrate that FACT likely has a role in heterochromatin silencing.
One of the key questions in the epigenetic field is how epigenetically determined chromatin states, such as heterochromatin, are inherited during the S phase of the cell cycle. Most heterochromatin research has focused on the protein components of heterochromatin, such as Sir proteins in S. cerevisiae, Swi6 in S. pombe, HP1 in higher eukaryotic cells, as well as the underlying histone modifications. We summarize findings from different organisms regarding the potential roles of many factors known to be involved in DNA replication and DNA RC nucleosome assembly and how they impact on heterochromatin silencing and inheritance. While how each of these DNA replication proteins functions in heterochromatin silencing is not clear, we suspect that these proteins likely impact on heterochromatin silencing in one of three ways (Fig. 3). First, these proteins facilitate DNA replication and nucleosome assembly of heterochromatin, which in turn impacts on heterochromatin formation and inheritance ‘indirectly’. Second, these proteins recruit factors important for establishing and maintaining heterochromatin silencing and/or modifications at heterochromatin. Third, these proteins coordinate with siRNA machinery to maintain heterochromatin and ensure inheritance. Future studies are needed to better understand how these proteins promote heterochromatin formation and silencing during S phase of the cell cycle.
In addition to the factors we discussed above, the ATP-dependent chromatin-remodeling machines, which alter DNA-histone interactions in ATP-dependent manner and are known to be critical in gene transcription , are likely to be involved in heterochromatin replication/maintenance. For instance, ACF1 (ATP-utilizing chromatin assembly and remodeling factor 1) and an ISWI isoform, SNF2H (sucrose nonfermenting-2 homolog), are specifically enriched in replicating pericentromeric heterochromatin . In budding yeast, three chromatin remodelers including Isw1, Snf2, and Fun30 are involved in transcriptional silencing at distinct heterochromatin regions [145–148]. More recently, mammalian SMACARD1, the homolog of yeast Fun30, was identified to be co-purified with transcription repressor KAP-1, histone deacetylases HDAC1/HDAC2, and histone methyltransferase G9a/GLP. The α-thalassemia X-linked mental retardation protein (ATRX) is required for the formation of pericentric heterochromatin in higher eukaryotes, possibly working with histone chaperone Daxx for deposition of H3.3 to telomeric heterochromatin regions [134,149–151]. These results are consistent with the idea that duplication of heterochromatin is much more complex than we previously imagined. Although many pieces of the puzzle are still missing, exciting working models regarding how various chromatin states are specified and passed on to the next generation begin to be established [152,153]. For instance, after reviewing possible models for epigenetic inheritance, Moazed  proposed that non-coding RNAs and DNA-sequence-specific binding proteins are likely to play an important role in specifying chromatin regions for inheritance. It is not clear as to how small RNAs and their processing machinery communicate with DNA replication factors for inheritance of chromatin domains. Recently, it has been shown that DNA replication proteins, such as Cdc20, serve as links between DNA replication and siRNA generation . However, precisely how proteins involved in DNA replication coordinate with RNA production and reestablishment of histone marks remains largely unknown. We look forward to future experiments identifying and charactering macromolecular complexes connecting these processes, yielding mechanistic insight into how various chromatin states are specified and passed on to the next generation.
This work was supported by the grants from NIH (GM72719 and GM81838 to Z.Z). Z.Z. is a scholar of the Leukemia and Lymphoma Society.
We thank Rebecca Burgess for her editing on the manuscript. We apologize to those whose publications have not been cited in this review due to space limit.