Báo cáo Y học: Chromatin assembly Cooperation between histone chaperones and ATP-dependent nucleosome remodeling machines docx

MINIREVIEW Chromatin assembly Cooperation between histone chaperones and ATP-dependent nucleosome remodeling machines Jessica K. Tyler Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Health Sciences Center, Denver, CO, USA Chromatin is a highly dynamic structure that plays an essential role in regulating all nuclear processes that utilize the DNA template including DNA repair, replication, transcription and recombination. Thus, the mechanisms by which chromatin structures are assembled and modified are questions of broad interest. This minireview will focus on two groups of proteins: (a) histone chaperones and (b) ATP- dependent chromatin remodeling machines, that co-operate to assemble DNA and histone proteins into chromatin. The current understanding of how histone chaperones and ATP- dependent remodeling machines coordinately assemble chromatin in vitro will be discussed, together with the growing body of genetic evidence that supports the role of histone chaperones in the cell. Keywords: chromatin; histone chaperone; nucleosome remodeling; acetylation; DNA replication; DNA repair; CAF-1; ACF; ASF1; Rad53. INTRODUCTION TO CHROMATIN The eukaryotic genome is packaged into a nucleoprotein structure known as chromatin. The basic repeating unit of chromatin, the nucleosome core particle, comprises approximately two turns of DNA wrapped around two molecules of each core histone protein; H2A, H2B, H3 and H4 ([1]; and reviewed in [2]). Nucleosomes are regularly spaced along eukaryotic DNA with a repeat length (i.e. center-to-center internucleosomal distance) of 180–200 bp, which probably reflects the most energetically favorable arrangement [3]. The regular nucleosomal arrays comprise a 10-nm fiber of chromatin and folds into a 30-nm fiber upon the incorporation of a single molecule of linker histone, such as histone H1, per nucleosome. Higher levels of chromatin compaction involve additional proteins, many of which are unknown, in order to achieve the astounding degree of condensation required to yield mitotic chromosomes. Histone proteins are highly conserved among eukaryotic organisms. Consistent with this fact, the recently published crystal structure of the yeast nucleosome is surprisingly similar to the metazoan nucleosome structure [1,4]. Even some archaebacteria contain nucleosomes comprised of 80 bp of DNA wrapped around two molecules each of the two archaeal histones [5]. The similarity of nucleosome structures among eukaryotic organisms indicates that the mechanism of chromatin assembly is likely to be highly conserved across all eukaryotes. As such, we can combine insight provided from systems as diverse as yeast, Dro- sophila, Xenopus and humans in order to build our understanding of the machinery and mechanism of chro- matin assembly. The main players in chromatin assembly that have been identified to date are the histone chaperones and a protein complex that remodels nucleosomes in an ATP- dependent manner. This minireview focuses on their identification and role in chromatin assembly in vitro, and the evidence that the histone chaperones assemble chromatin in vivo. Other aspects of chromatin assembly, including cell cycle regulation of histone chaperone function and their role in epigenetic inheritance are covered in a series of recent reviews and will not be addressed here [6–11]. CHROMATIN ASSEMBLY IN THE CELL Little is known about how chromatin is assembled in the cell. We do know that the majority of chromatin assembly occurs immediately following DNA replication, where nucleosomes are disrupted by the passage of the replication machinery (reviewed in [2,7]). The naked daughter strands of newly replicated DNA appear to be rapidly assembled into chromatin by a multistep process that involves the initial deposition of histones H3 and H4 followed by incorporation of two histone H2A-H2B dimers to complete Correspondence to J. Tyler, Department of Biochemistry and Molecular Genetics, School of Medicine B121, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver CO 80262, USA. Fax: + 1 303 3155467, Tel.: + 1 303 3158163, E-mail: Jessica.tyler@uchsc.edu Abbreviations: ACF, ATP-utilizing chromatin assembly and remodeling factor; ASF1, antisilencing function 1; CAC1, chromatin assembly complex 1; CAF-1, chromatin assembly factor 1; CHRAC, chromatin accessibility complex; ISWI, imitation switch; NAP-1, nucleosome assembly protein 1; NURF, nucleosome remodeling factor; PCNA, proliferating cell nuclear antigen; RSF, remodeling and spacing factor. Dedication: This Minireview Series is dedicated to Dr Alan Wolffe, deceased 26 May 2001. (Received 8 October 2001, revised 21 November 2001, accepted 29 November 2001) Eur. J. Biochem. 269, 2268–2274 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02890.x the nucleosome. The parental nucleosomes disassemble into H3-H4 tetramers and H2A-H2B dimers and are then distributed, by an unknown mechanism, randomly between the two daughter DNA duplexes [12]. However, the parental chromatin only provides half of the histones that are required; the remainder is synthesized de novo during S phase of the cell cycle [13]. The first step in the assembly of chromatin is the nuclear import of the newly synthesized core histones. This is mediated by a network of soluble transport receptors called karyopherins (Kaps, also called importins, exportins, and transportins) that bind to the nuclear localization signal in the N-terminal domain of each core histone [14,15]. These newly synthesized histones are acetylated at lysine residues 5 and 12 of H4, and lysine residue 14 of H3 (in Drosophila) prior to their assembly into chromatin, and are rapidly deacetylated after their deposition onto newly replicated DNA [16,17]. The functional relevance for this specific pattern of histone acetylation and subsequent deacetylation during chroma- tin assembly is not understood. In vivo,theN-terminal domains of histone H3 and H4 are functionally redundant for chromatin assembly [18]. Furthermore, in the absence of the N-terminal domain of histone H3, acetylation of only lysine 5, 8, or 12 of histone H4 is required for chromatin assembly [19]. The acetylation of histone N-terminal domains is not required for the deposition of histones onto the DNA, but has recently been shown to facilitate the regular spacing of nucleosomes during chromatin assembly in vitro [20]. CHROMATIN ASSEMBLY IS MEDIATED BY HISTONE CHAPERONES Key insight into the mechanism of chromatin assembly in the cell has been provided by biochemical studies of histone–DNA interactions. Histone proteins are rich in positively charged basic amino acids that have an intrinsic affinity for the negatively charged phosphate groups in DNA; mixing histones and DNA in vitro at physiological salt concentration leads to the rapid formation of undefined insoluble aggregates (Fig. 1A). However, the presence of additional anionic factors that shield the charge of the histones from DNA, allow chromatin assembly to occur in a regulated and ordered manner (Fig. 1B). It would appear that additional negatively charged molecules can act as histone chaperones to allow H3-H4 tetramers to bind to DNA first, due to their higher affinity for DNA as compared to H2A-H2B dimers (Fig. 1B). The subsequent deposition of H2A-H2B dimers is likely to be driven by the higher affinity of H2A-H2B for subnucleosomal particles comprising DNA and H3-H4, as compared to their affinity for either a histone chaperone or DNA [21]. As such, chromatin assembly that is mediated by histone chaperones in vitro mimics the stepwise process that occurs in the cell. Using biochemical approaches to search for physiolog- ically relevant histone chaperones has led to the identifica- tion of numerous molecules that can bind to histone proteins and facilitate chromatin assembly in vitro (reviewed in [22,23]). Unfortunately, this area of research has been fraught with artifacts due to the permissive nature of the Fig. 1. Model for the role of histone chaperones and remodeling machines in chromatin assem- bly. (A) Positively charged histone proteins and negatively charged (Ô–veÕ) DNA molecules have a high affinity for each other and rapidly form insoluble aggregates when mixed in vitro. (B) Histone chaperones (defined here as any negatively charged molecule that can bind to histone proteins and facilitate nucleosome formation) are likely to function by transiently shielding the charge of the positively charged histones from the negatively charged DNA molecules. Histone H3-H4 tetramers have a higher affinity for DNA (as indicated by the ++), as compared to histone H2A-H2B dimers (+); this may be sufficient to facilitate nucleosome formation in a step-wise manner inthepresenceofahistonechaperone.Other histone chaperones may bind to a preformed histone octamer and transfer it to DNA. (C) ATP-dependent chromatin remodeling machines (such as ACF) are required in order to generate regular physiological spacing of nucleosomes in vitro. In one report, the remodeling machine was additionally required for formation of the nucleosome [27]. Ó FEBS 2002 Mechanism of chromatin assembly (Eur. J. Biochem. 269) 2269 biochemical chromatin assembly systems that were used. Almost any molecule that could shield the basic charge of histone proteins from DNA, including pectin, RNA, polyglutamic acid and even salt, was found to function as a histone chaperone in vitro with little or no relevance for the assembly of chromatin in vivo. However, the use of cell-free chromatin assembly systems coupled to ongoing DNA replication has been more productive in identifying histone chaperones in vitro that also appear to assemble chromatin in the cell, as discussed later. ATP-REMODELING IS INTRINSIC TO CHROMATIN ASSEMBLY Histone chaperones are not sufficient to generate regular arrays of nucleosomes with 180–200 bp spacing in vitro. Instead, histone chaperones lead to the assembly of irregularly spaced or closely spaced nucleosome arrays in vitro (Fig. 1B). Presumably, factors in addition to histone chaperones are required in order to generate the physiolog- ically spaced arrays of nucleosomes that are seen in the cell. Indeed, it has long been known that chromatin assembly in crude extracts requires ATP hydrolysis in order to generate regular arrays of physiologically spaced nucleosomes [24]. Accordingly, biochemical fractionation of crude Drosophila embryo extracts (a rich source of chromatin assembly factors due to the rapid rounds of DNA replication and concomitant chromatin assembly that occur during early embryogenesis) identified a second key component of the chromatin assembly machinery: an ATP-dependent chromatin remodeling factor (reviewed in [25]). This ATP-dependent chromatin remodeling factor was found independently by two groups and was termed ACF (for ATP-utilizing chromatin assembly and remodeling factor) and CHRAC (for chromatin accessibility complex) [26–29]. The catalytic component of ACF is the Drosophila imitation switch (ISWI) ATP-ase protein. ISWI is a component of at least one other distinct Drosophila protein complex, called NURF, that mediates ATP-dependent remodeling of nucleosomes during transcriptional activation in vitro [25]. The other component of ACF, Acf1, enhances and modulates the activity of ISWI during chromatin assembly and nucleosome sliding [27,29]. The CHRAC complex has two additional low molecular mass proteins, as compared to ACF, that are not required for chromatin assembly in vitro [29,30]. ACF can therefore be viewed as the functional core of CHRAC. It is not known whether ACF mediates chromatin assembly in the cell. Regular arrays of nucleosomes are generated with a combination of a histone chaperone and ACF in an ATP-dependent manner in vitro (Fig. 1C [26,27]). The energy provided by ATP-hydrolysis presumably enables the many histone–DNA interactions in the nucleosome to transiently break, allowing the histone octamer to move along the DNA until regular spacing between nucleosomes is achieved. This nucleosome remodeling process is likely to require large amounts of ATP hydrolysis, but the benefit to the cell for generating regularly spaced nucleosomes is unknown. In addition to its role in ATP-dependent nucleosome spacing, ACF facilitates histone chaperone-mediated deposition of histones onto the DNA [27]. The assembly of regular arrays of nucleosomes with physiological spacing in vitro appears to require close cooperation between histones, DNA, ACF and a histone chaperone. Similar protein complexes to Drosophila ACF have since been identified in humans and Xenopus [31–34]. However, a human counterpart called the remodeling and spacing factor (RSF) exhibits some striking functional distinctions from ACF. Purified RSF is sufficient to deposit histones and space nucleosomes during chromatin assembly, in the absence of a histone chaperone [20]. In addition, each RSF molecule only assembles one DNA molecule into chroma- tin, whereas each ACF molecule can assemble multiple DNA molecules into chromatin [20,27]. These differences may be due to the ability of RSF to bind to histones H3-H4 and simultaneously act as a histone chaperone and a remodeling factor [20]. IDENTIFICATION OF HISTONE CHAPERONES THAT MEDIATE DNA REPLICATION-COUPLED CHROMATIN ASSEMBLY The majority of chromatin assembly is tightly coupled to DNA replication in the cell. Accordingly, chromatin assembly systems that preferentially assemble chromatin onto replicating DNA templates have been developed using crude protein extracts derived from Xenopus oocytes and human cells [35,36]. This replication-dependent chromatin assembly occurs in a stepwise manner in vitro, mirroring the stepwise assembly of chromatin onto newly replicated DNA observed in the cell (Fig. 2; [35,37]). Fractionation of the human cell extract identified a heterotrimeric protein complex termed chromatin assembly factor-1 (CAF-1) whose structure and sequence has been conserved among eukaryotic organisms (reviewed in [8–11]). CAF-1 copurifies with histones displaying a similar pattern of lysine acetyla- tion to that reported for newly synthesized H4 [38]. There- fore, CAF-1 appears to act as a histone chaperone to deposit newly synthesized histones H3 and H4 onto newly replicated DNA in vitro (Fig. 2; [39]). The assembly of chromatin by CAF-1 occurs by a mechanism that is preferentially coupled to DNA synthesis; this coupling is likely to be at least partly mediated via interactions between CAF-1 and the PCNA component of the DNA replication machinery that may serve to localize CAF-1 to sites of DNA synthesis (Fig. 3; [40,41]). CAF-1 may be a physiologically relevant histone chaperone as it localizes to sites of ongoing DNA replication in the cell and is required for efficient chromatin-mediated transcriptional silencing (reviewed in [8,9,11]). However, yeast disrupted for CAF-1 have no growth defects, indicating there are likely to be additional histone chaperones in the cell. A novel histone chaperone, termed replication-coupling assembly factor (RCAF), was identified by its ability to facilitate CAF-1- mediated assembly of nucleosomes onto newly replicated DNA in vitro [42]. RCAF is a complex of the Drosophila homologue of the yeast antisilencing function 1 (ASF1) protein and histones H3 and H4 [42–44]. The histones that are associated with ASF1 have an acetylation pattern characteristic of newly synthesized histones. It appears therefore, that ASF1 is a histone chaperone for newly synthesized histones H3 and H4 in vitro. However, ASF1 does not have a preference for assembling newly replicated DNA into chromatin in vitro in the absence of CAF-1 2270 J. K. Tyler (Eur. J. Biochem. 269) Ó FEBS 2002 [42,45,46]. It has recently been shown that ASF1 binds directly to CAF-1 in vivo and in vitro [47]. Therefore, the ability of ASF1 to facilitate chromatin assembly preferen- tially onto newly replicated DNA in vitro is likely mediated by CAF-1 targeting ASF1 to the DNA replication fork (Fig. 3; [47]). Genetic analyses in yeast support the biochemical evidence that ASF1 is a histone chaperone that functions during the assembly of newly synthesized DNA into chromatin [42–44]. Although ASF1 is not an essential gene in budding yeast, asf1 mutants grow slowly due to an elongated G 2 /M phase of the cell cycle; this is extended to an apparent arrest in G 2 /M phase under various conditions of stress [42–44]. The sensitivity of asf1-mutant yeast to hydroxyurea (a reagent that depletes the endogenous nucleotide pools in the cell) indicates that under conditions when DNA synthesis is compromised, ASF1 may be required in order to generate chromosomes that are com- petent to pass through G 2 /M phase. The delay in progres- sion through G 2 /M phase of asf1 mutants is reminiscent of a problem observed in histone H4 mutants lacking acetylat- able lysines [48,49] and in conditional mutants of compo- nents of the NuA4 histone acetyltransferase complex [50,51]. Taken together, these studies suggest that ASF1 is a histone chaperone for newly synthesized and specifically acetylated histones H3-H4. Initial studies indicated that ASF1 and CAF-1 are both histone chaperones for newly synthesized histones H3-H4 in vitro. Yet the assembly of newly replicated DNA into chromatin depends on both ASF1 and CAF-1 in vitro,at least for the Drosophila and yeast factors [42,46]. The codependence of CAF-1 and ASF1 during the assembly of newly replicated DNA into chromatin was not previously detected due to the endogenous ASF1 in the human cell free extract used to study CAF-1 function. Genetic analyses, however, have not re-affirmed the codependence between CAF-1 and ASF1 during the assembly of newly replicated DNA into chromatin [42]. Budding yeast disrupted for ASF1 have many phenotypesthatare not shared with CAF-1 mutants, including growth defects, sensitivity to double- strand DNA damage, sensitivity to hydroxyurea, suppres- sor of Ty or Spt phenotype, and disrupted histone gene expression [42–44,46,52]. While other roles of ASF1 and CAF-1 appear to be overlapping, sensitivity to UV-irradi- ation and transcriptional silencing defects are enhanced in yeast that are mutant for both CAF-1 and ASF1, as compared to yeast strains that are mutant for only CAF-1 or ASF1 [42]. Taken together, the genetic evidence indicates that ASF1 has roles that are not shared with CAF-1, as well as roles that are redundant with CAF-1 in the cell. However, the physical interaction between ASF1 and CAF-1 in vivo [47] indicates that the codependence between ASF1 and CAF-1 is probably not an in vitro artifact. Instead, there may be additional histone chaperones in vivo that are functionally redundant with ASF1 and/or CAF-1 that are either missing or inactive in the cell-free assembly system. WHAT ARE THE ADDITIONAL HISTONE CHAPERONES? There are probably additional histone chaperones in the cell, as yeast lacking both the ASF1 and CAF-1 histone chaperones are viable [42]. No sequence motifs have yet been identified to indicate a potential function as a histone chaperone. Therefore, we have had to rely on biochemical fractionation and genetic analyses in order to identify novel histone chaperones. Biochemical approaches have identified Fig. 2. Model for chromatin assembly at the DNA replication fork. In vivo studies have indicated that the DNA replication machinery (large red oval) displaces all the histones from the parental chromatin. The two daughter DNA duplexes are rapidly assembled into chro- matin in a stepwise manner from a mixture of old histones and de novo synthesized histones. H3-H4 tetramers (large yellow-coloured ovals) are deposited first, followed by two H2A-H2B dimers (small cream- coloured ovals) to complete the nucleosome. The newly synthesized H3 and H4 proteins have a specific conserved pattern of acetylation in the cell (indicated by ÔAcÕ) and are deacetylated soon after nucleosome formation. Also indicated are chromatin assembly factors that have been identified biochemically and may function to mediate chromatin assembly in vivo: histone chaperones for newly synthesized H3 and H4 (ASF1 and CAF-1) and for H2A-H2B (NAP-1). ATP-hydrolysis is required for chromatin assembly, and may reflect a role for the ATP- dependent chromatin remodeling factor ACF in the cell. Linker histones (small red-coloured ovals) can be incorporated into nucleo- seomes at a later stage of chromatin assembly, as mediated by an unknown chaperone in the cell. Fig. 3. Model to explain the biochemical requirement of ASF1 for the assembly of newly replicated DNA into chromatin. Assembly of newly replicated DNA into chromatin requires both ASF1 and CAF-1 in vitro. CAF-1 is recruited to the DNA replication fork via its inter- action with the PCNA component of the replication machinery (yellow rings). The recently identified interaction between ASF1 and CAF-1 may in turn target ASF1 to the DNA replication fork. Ó FEBS 2002 Mechanism of chromatin assembly (Eur. J. Biochem. 269) 2271 the H2A-H2B binding protein nucleosome assembly protein 1 (NAP-1), which is highly conserved among eukaryotic species [53–56]. NAP-1 in Drosophila and the closely related human protein NAP-2 function as histone chaperones in vitro [56,57]. Furthermore, Drosophila NAP-1 and human NAP-2 bind to histone proteins H2A-H2B in vivo and move from the cytoplasm to the nucleus as cells progress from G 1 to S phase concomitant with the activation of nucleosome assembly during DNA replication [56,57], although NAP-1 is not required for the nuclear import of H2A-H2B [14]. The function of NAP-1 during chromatin assembly in vivo is likely to be redundant with still unknown histone chaper- ones; yeast containing disrupted ASF1, CAC1 (encoding the largest subunit of CAF-1), and NAP-1 showed no greater growth defects or transcriptional silencing defects than ASF1, CAC1 double mutants (R. Kamakaka, National Institutes of Health, Bethesda, MD, USA, personal communication). It is interesting to note that budding yeast can tolerate loss of individual histone chaperones much more than higher eukaryotes. For example, whereas ASF1 mutants are viable in yeast, ASF1 is an essential gene product in Drosophila (Y. Moshkin, J. Kenisson & F. Karch, Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland, personal communica- tion). The ability of yeast to survive without ASF1 may reflect the fact that yeast has a more open chromatin structure than higher eukaryotes, which in turn may be related to the subtle destabilization of the structure of the yeast core nucleosome particle as compared to that of metazoans [4]. In addition, it suggests that higher eukar- yotes may be subject to extra levels of regulation of their nuclear functions by chromatin structure. DNA REPAIR MAY REQUIRE HISTONE CHAPERONES DNA repair is the second major site of DNA synthesis in the cell, and it may also entail concomitant chromatin assembly. Accordingly, CAF-1 has been implicated in the repair of UV-induced DNA damage. Yeast disrupted for CAF-1 function are sensitive to UV irradiation [58]. Furthermore, CAF-1 stimulates chromatin assembly fol- lowing nucleotide excision repair in vitro [59] and is recruited to chromatin following UV irradiation in vivo [60]. Pheno- typic analyses of yeast with disrupted ASF1 have indicated that ASF1 also plays a role in the response to UV irradiation that is apparent in the absence of CAF-1 [42]. More strikingly, budding yeast with disrupted ASF1 are highly sensitive to drugs that generate double-strand DNA breaks, indicating that ASF1 may play a role in double- strand repair [42–44]. Recent studies have indicated that the chromatin assem- bly function of ASF1 is a novel target of the DNA damage response [61,62]. The DNA damage checkpoint allows cells to respond to DNA damage or replicative stress by activating DNA repair processes and arresting cells until the damage is repaired [63]. In the absence of DNA damage or replicative stress, nearly all the soluble pool of the Rad53 DNA damage checkpoint protein in yeast is bound to ASF1 [61,62] (Fig. 4A). The interaction between Rad53 and ASF1 appears to prevent ASF1 from binding to histones and assembling chromatin [61]. The release of ASF1 from Rad53 correlates with the DNA damage- or replicative stress-stimulated phosphorylation of Rad53, allowing ASF1 to bind to histones and assemble chromatin [61] (Fig. 4B). Furthermore, over-expression of ASF1 suppresses the defects that result from the deletion of RAD53 [62]. One model that is consistent with the available data is that activation of Rad53 by the DNA damage checkpoint releases ASF1 into the vicinity of the DNA lesion in order to contribute to the assembly of the newly synthesized DNA into chromatin (Fig. 4B). THIS IS JUST THE BEGINNING We are only now gaining real insight into the machinery and mechanism of chromatin assembly. Chromatin assembly requires a negatively charged histone chaperone in order to shield the basic histones from the acidic DNA, and an ATP- dependent chromatin remodeling factor, ACF, that facili- tates the formation of nucleosomes and their regular spacing along the DNA. However, many questions remain unanswered. For example, what is the identity of the other histone chaperone(s) for H3-H4, H2A-H2B and H1 in the cell? How do histone chaperones and ATP-remodeling machines function together? How are specific chromatin structures re-established onto the daughter DNA duplexes so as to maintain patterns of programmed gene expression through multiple generations? What is the role of histone chaperones in DNA repair? The answers to these questions will provide important insight not only into the mechanism of chromatin assembly, but also insight into the influence of Fig. 4. Model for the regulation of ASF1 by the DNA damage check- point machinery. (A)Rad53isboundtoASF1intheabsenceofan activated DNA damage checkpoint, preventing ASF1 from binding histones and assembling chromatin. (B) Following activation of the DNA damage checkpoint, Rad53 becomes phosphorylated. ASF1 is released from the phosphorylated Rad53, where it can then bind to histones and assemble chromatin. 2272 J. K. Tyler (Eur. J. Biochem. 269) Ó FEBS 2002 chromatin assembly on gene expression, DNA replication, repair, recombination, growth and development. ACKNOWLEDGEMENTS I would like to thank F. Karch and R. Kamakaka for sharing results prior to publication. I am highly grateful to Les Krushel, Josh Ramey, Jeff Linger and Susan Howar for critical reading of this manuscript. REFERENCES 1. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. & Richmond, T.J. (1997) Crystal structure of the nucleosome core particle at 2.8 A ˚ resolution. Nature 389, 251–260. 2. Wolffe, A.P. (1998) Chromatin Structure and Function.Academic Press, San Diego. 3. 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MINIREVIEW Chromatin assembly Cooperation between histone chaperones and ATP-dependent nucleosome remodeling machines Jessica K. Tyler Department of Biochemistry and Molecular Genetics,. DNA REPLICATION-COUPLED CHROMATIN ASSEMBLY The majority of chromatin assembly is tightly coupled to DNA replication in the cell. Accordingly, chromatin assembly systems that preferentially assemble chromatin onto. chromatin assembly factors that have been identified biochemically and may function to mediate chromatin assembly in vivo: histone chaperones for newly synthesized H3 and H4 (ASF1 and CAF-1) and for