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The ins and outs of nucleosome assembly Jill A Mello* and Geneviève Almouzni† De novo nucleosome assembly coupled to DNA replication and repair in vitro involves the histone chaperone chromatin assembly factor 1 (CAF-1). Recent studies support a model in which CAF-1 can be targeted to newly synthesized DNA through a direct interaction with proliferating cell nuclear antigen (PCNA) and can act synergistically with a newly identified histone chaperone. Insights have also been obtained into mechanisms by which this CAF-1-dependent pathway can establish a repressed chromatin state. Addresses Institut Curie, Research section, UMR 218 du Centre National de la Recherche Scientifique (CNRS), 75248 Paris cedex 05, France *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Genetics & Development 2001, 11:136–141 0959-437X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ACF ATP-utilizing chromatin assembly and remodeling factor ASF1 anti-silencing factor 1 CAF-1 chromatin assembly factor 1 CHRAC chromatin accessibility complex HP1 heterochromatin-binding protein 1 ISWI SWI2-related imitation switch NAP-1 nucleosome assembly protein 1 PCNA proliferating cell nuclear antigen
Introduction Chromatin plays a critical role in the regulation of DNA metabolic processes, including replication, repair and gene expression. Efficient assembly of nucleosomes — the fundamental repeating units of chromatin — onto newly synthesized DNA is thus essential for maintaining proper genome function. At the replication fork, parental nucleosomes are redistributed between the two nascent chromatids, and de novo nucleosome assembly provides the required nucleosome complement. Evidence indicates that a de novo pathway also operates during the repair of DNA damage. Here we discuss recent insights into the mechanism of de novo nucleosome assembly and how the requisite proteins may participate to impart a transcriptional state.
Nucleosome assembly and histone chaperones: a coordinated network? Nucleosome assembly follows a two-step process in which a tetramer of histones H3 and H4 is first deposited onto DNA, and subsequently two H2A–H2B heterodimers are deposited to yield an octamer core around which 146 base pairs (bp) of DNA is wrapped. The ordered assembly of nucleosomes within the nucleus is thought to be mediated by histone chaperones [1,2] — proteins that bind and effectively neutralize highly charged histones, thereby preventing their nonspecific aggregation with DNA. Several histone chaperones have been identified by using in vitro
reconstitution experiments, and a current challenge is to understand how they function — either alone or in a coordinated manner — in the assembly of nucleosomes. The best-characterized histone chaperone is chromatin assembly factor 1 (CAF-1) — a multisubunit protein, which in humans contains the three subunits p150, p60 and p48, whose function is evolutionarily conserved [3]. CAF-1 is associated with newly synthesized forms of H3 and H4. The most striking feature of CAF-1 is its unique ability to promote preferentially nucleosome assembly onto newly synthesized DNA, either during replication [4,5] or during nucleotide excision repair [6] in vitro; this makes CAF-1 a strong candidate for a coordinator of nucleosome assembly during DNA synthesis in vivo. The localization of CAF-1 to replication foci during S phase, at both euchromatin and heterochromatin regions [7,8•], and its recruitment to chromatin in G1- or G2-phase cells that have undergone UV DNA damage [9] strongly support this idea. Null mutants of CAF-1 homologues in Saccharomyces cerevisiae are mildly sensitive to UV radiation but are otherwise perfectly viable [10,11•], suggesting that alternate de novo assembly pathways are operative, at least during S phase, in this organism. Mutants of CAF-1 in budding yeast also show defects in transcriptional silencing at telomeres and at the mating-type loci HMLα and HMRa [10,12–14], indicating that CAF-1 is involved in maintaining heterochromatin at these loci. In mammalian cells, an interaction between CAF-1 and heterochromatin-binding protein 1 (HP1) may be of possible relevance [15]. To clarify further the general role of CAF-1 in nucleosome assembly coordinated to DNA synthesis, both during and outside S phase, it will be critical to assess its in vivo significance in higher eukaryotes. It will also be important to understand why certain chromatin regions are particularly stringent in their requirement for CAF-1. The apparent existence in budding yeast of redundant de novo assembly pathways has spurred the search for new assembly factors. One outcome is the recent purification of a new nucleosome assembly complex, RCAF, from Drosophila embryo extracts; RCAF was identified by its ability to synergize with CAF-1 to promote nucleosome assembly during DNA replication in vitro. RCAF comprises the highly evolutionarily conserved protein anti-silencing factor-1 (ASF1), of which the human homologue displays histone chaperone activity [16], together with newly synthesized forms of H3 and H4 [17••]. Mutants of Asf1, either alone or in combination with mutants of CAF-1, are viable in yeast, which indicates that other assembly pathways exist. When compared with CAF-1, however, Asf1 mutants display additional growth defects and
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Figure 1 A model for targeting nucleosome assembly to DNA transactions. De novo nucleosome assembly involves the deposition of a tetramer of newly synthesized forms of histones H3 and H4 in a process involving the histone chaperone CAF-1, followed by the addition of two H2A–H2B heterodimers. The specificity for targeting nucleosome assembly to DNA transactions is achieved through a direct interaction of CAF-1 with PCNA, either at sites of DNA repair (left) or at the replication fork (right). In addition, we present the hypothesis that another histone chaperone, ASF1, may deliver newly synthesized H3 and H4 to DNA targeted through CAF-1, either by: (i) shuttling the histones to CAF-1 prior to its interaction with PCNA or (ii) directly at sites of replication or repair. Alternatively, ASF1 may deliver H3 and H4 in a distinct, CAF-1 independent manner.
(H3–H4)2 ASF1 (i) (ii)
2 X H2A–H2B
(ii) (H3–H4)2 CAF-1 CAF-1
CAF-1
PCNA PCNA DNA repair site
exhibit sensitivity to a broader range of DNA-damaging agents [17••,18]. Furthermore, whereas a defect in Asf1 shows very little effect on silencing at telomeres or mating-type loci, a double mutant of CAF-1 and Asf1 displays more pronounced silencing defects than does either single mutant [17••,18,19]. From these studies, we can infer that Asf1 and CAF-1 are to some degree functionally distinct, and that both participate in heterochromatic silencing. Further clues to the role of Asf1 are awaited eagerly. We are tempted to speculate, however, that Asf1 may act as a histone donor for CAF-1, shuttling H3–H4 to CAF-1 that is localized at sites of assembly. Indeed, the general idea that chaperones might function within a coordinated assembly line to deliver histones to target DNA is an attractive strategy for achieving a fast, efficient process. Moreover, introducing specialized chaperones, which could perhaps be associated with specifically modified histones or histone variants, into a chaperone network might be a way to achieve temporal or regional control for establishing specialized chromatin structures. If we consider that nucleosome assembly is achieved through the function of a coordinated network, it is likely that the deposition of H2A–H2B dimers on the H3–H4 tetramer is also mediated by a histone chaperone. Nucleosome assembly protein 1 (NAP-1) is an evolutionarily conserved histone chaperone that is associated with H2A–H2B both in human cells [20] and in Drosophila embryo extracts [21]. Both Drosophila NAP-1 and the highly related human protein NAP-2 [22] undergo rapid localization to the nucleus in S phase, suggesting that they have a role in DNA replication. But, in a manner independent of replication, NAP-1 can promote nucleosome assembly in the presence of all core histones [23] and can cooperate with ATP-utilizing chromatin assembly and remodeling factor (ACF) to assemble regularly spaced
Replication fork Current Opinion in Genetics & Development
nucleosomal arrays [24,25•]. Thus if NAP-1 does play a role in de novo nucleosome assembly, it may rely on the initial specificity of CAF-1 or other chaperones for newly synthesized DNA in the first step of the process. In this regard, the recently reported ability of CAF-1 p60 to interact with H2A and H2B [26•] presents the possibility that NAP-1 or other factors might deliver these histones to CAF-1 localized at sites of DNA synthesis, which might, in turn, facilitate their deposition through p60. In addition to the idea that chaperones might cooperate as part of a network, it is important to stress the non-mutually exclusive possibility that some chaperones might possess a dedicated function. The histone chaperones nucleoplasmin-1 and N1/N2 are associated with H2A–H2B and H3–H4, respectively [1]. To date, these proteins have been identified only in oocytes of Xenopus and Drosophila, and may function exclusively during the rapid cell divisions required during early embryogenesis. Interestingly, a recent study in mice of the X-linked NAP-1 gene family member, Nap1l2, has shown that its encoded protein is cytoplasmic in G1 but localizes to chromatin during S phase, and is expressed mainly in neurons [27•]. In addition, inactivation of Nap1l2 was lethal from mid-gestation, but caused severe defects in neuronal cell proliferation in surviving mutant chimeric embryos [27•]. These results suggest that Nap1/2 may be a tissue-specific chromatin factor. Thus, it is reasonable to speculate that other tissue-specific chaperones exist and may have dedicated roles during development.
Targeting nucleosome assembly to DNA transactions Just as we have begun to understand how molecular chaperones mediate the deposition of histones onto newly synthesized DNA, light has also been recently shed on another critical question: how are these chaperones brought to the sites of DNA transactions? The remarkable
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specificity of CAF-1 for nucleosome assembly on replicated and repaired DNA in vitro has been explained recently by its ability to recognize DNA that is topologically marked by the presence of the proliferating cell nuclear antigen (PCNA) [28••,29••]. PCNA is a homotrimeric protein that forms a sliding clamp around DNA and functions as a DNA polymerase processivity factor during replication and nucleotide excision repair [30]. In vivo, co-immunolocalization of PCNA and CAF-1 at replication foci and to chromatin of UV-irradiated cells strongly supports a role for PCNA as a recruitment factor for chromatin assembly machinery [9,28••]. At a biochemical level, the human CAF-1 p150 subunit interacts directly with PCNA through contacts with the interdomain connecting loop on the outer, front side of the PCNA ring — a region known to interact with several other proteins [26•,29••]. Indeed, PCNA interacts with many proteins [31], and a picture is beginning to emerge in which PCNA acts as a central coordinator for replication, repair, epigenetic inheritance and cell-cycle control. A more detailed description of how p150 and other interactor proteins compete for binding to PCNA, or alternatively are accommodated by the homotrimeric structure, would help to elucidate how several activities are coordinated at the replication fork and at repair sites. A PCNA-dependent chromatin assembly pathway targeting CAF-1 to DNA regions may have profound functional consequences, both in replication and repair. A recent study of PCNA mutants in S. cerevisiae has shown that, reminiscent of defects in CAF-1, several mutations in PCNA significantly decrease silencing at telomeres and at the mating-type HMR locus [26•]. This finding is consistent with the identified of a PCNA mutant in Drosophila which suppresses repression in the vicinity of heterochromatin [32,33]. All PCNA mutants that reduced silencing were also defective in the stable binding of the CAF-1 p150 subunit to chromatin, reinforcing the importance of their physical interaction in establishing specialized chromatin states. Interestingly, several PCNA mutations in combination with CAF-1 mutations displayed a synergistic decrease in silencing, suggesting that PCNA may also affect silencing in a manner at least partially independent of CAF-1, perhaps through the recruitment of other histone chaperones [26•]. In the context of replication, Shibahara and Stillman [28••] have proposed that asymmetric loading of PCNA on the leading and lagging strands at the replication fork may provide the opportunity to establish different chromatin structures on the two sister chromatids, thereby providing a mechanism for epigenetic inheritance. In the context of DNA repair, the specific chromatin structure established through this PCNA-dependent mechanism is, at this point in time, an open issue. It is possible that the PCNA-dependent targeting of chromatin assembly to DNA repair may set up a repressive chromatin structure, shutting down
DNA metabolism in the region until repair is complete, perhaps as part of a coordinated cell-cycle response to DNA damage [29••]. Compellingly, the repair and cellcycle checkpoint proteins Rad1/Rad9/Hus1 are predicted to form a PCNA-like heterotrimeric ring [34,35]. The possibility that CAF-1-dependent nucleosome assembly could be connected to this checkpoint pathway through a direct interaction between this complex and CAF-1 p150 deserves future investigation.
A cycle of histone acetylation and deacetylation and nucleosome assembly Newly synthesized histones H3 and H4 undergo a transient acetylation at their amino-terminal tails during S phase, before deposition onto replicated DNA, and are soon after deacetylated. In particular, newly synthesized H4 exhibits a highly conserved acetylation pattern at lysines 5 and 12 [36], and, importantly, this post-translational modification is distinct from the acetylation of chromatin-deposited histones associated with gene expression in euchromatin. The functional significance of post-translational H3–H4 acetylation for nucleosome assembly remains unknown, although it is clearly not required for core particle formation by dialysis in vitro. Beyond this, it has been suggested that the acetylation may be needed for nuclear import of histones or for their deposition by chromatin assembly factors. Addressing the latter possibility, a recent study has shown that CAF-1 can bind stably to tetramers of H3–H4 lacking their amino-terminal tails, and, moreover, can efficiently promote their preferential loading onto replicated DNA in vitro [37]. It nevertheless remains possible that acetylation is important for interaction with other histone chaperones, such as Asf1. An alternative role for H3–H4 acetylation might be in events that occur after deposition, either facilitating further chromatin maturation steps, or acting as a transient ‘marking’ mechanism for monitoring cell-cycle progression. In any case, it is noteworthy that in budding yeast, the type B histone acetyltransferase that modifies newly synthesized H4 is important for telomeric silencing [38], indicating functional significance for the acetylation of newly synthesized histones, at least in these regions. The deacetylation event that follows replication, like that of acetylation, is poorly understood. Rapid progress may come with the recent identification of several strong candidate enzymes for the deacetylation reaction [39•,40•]. In the genome, heterochromatin in particular exists in an underacetylated state. Of relevance, studies in Schizosaccharomyces pombe [41,42], and more recently in mammalian cells [43•], have shown that inhibition of deacetylation leads to delocalization of HP1, as well as to severe defects in centromeric heterochromatin regions and chromosome segregation. Thus, histone deacetylation seems to be critical for heterochromatin regions and for maintaining genomic integrity.
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At this point, it is worth recalling that in budding yeast the phenotype associated with mutants of CAF-1 is also revealed at heterochromatin regions. Interestingly, the smallest subunit of CAF-1, p48, associates with histone H4 and also with the catalytic subunit of the human deacetylase HD1 [3], suggesting a mechanism by which deacetylation might be tightly coupled to the assembly process. The fact that an observable phenotype associated with defects in many steps of the de novo nucleosome assembly pathway manifests primarily at heterochromatin regions may be understandable if we consider that within heterochromatin, deposition of acetylated H3–H4 and subsequent deacetylation appears to occur only one time during S phase [8•], whereas an overlapping dynamic acetylation/deacetylation process operates continuously in euchromatin. This situation would render heterochromatin more sensitive to changes introduced to the system.
Remodeling factors and nucleosome mobility Nucleosome assembly by histone chaperones alone is insufficient to obtain the long, regularly spaced nucleosomal arrays found in eukaryotic chromosomes, and requires the additional function of ATP-utilizing factors. Several factors have been purified from human cell, Drosophila embryo and Xenopus egg extracts on the basis of their ability to promote ATP-dependent nucleosome spacing, including ACF [24,44,45], the chromatin accessibility complex (CHRAC) [46,47], and the remodeling and spacing factor [48]. All remodeling factors identified to date contain a nucleosome-dependent ATP-utilizing subunit, many belonging to either the SWI/SNF or SWI2-related imitation switch (ISWI) chromatin remodelling families [49,50]. This ATPase subunit is thought to promote nucleosome spacing by facilitating nucleosome mobility, perhaps through a partial disruption of the nucleosome structure. The precise mechanism by which exact intranucleosomal spacing is determined, however, remains unclear. The ability of remodeling factors to disrupt histone–DNA interactions has been implicated clearly in the ‘opening up’ of the chromatin structure that occurs during transcription [51]. The extent to which nucleosomes are disassembled or remodeled at replication origins and sites of DNA repair remains to be determined, although it is clear that changes in chromatin structure occur [52]. Thus, it is equally important to learn how specific DNA regions within the chromatin architecture are made accessible to the replication or repair machinery, and it is conceivable that the ATP-dependent remodeling activities used in transcription may also be used in these contexts [53,54].
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which contains an ISWI-like ATPase, also contains two proteins related to the bacterial RuvB DNA helicase that catalyzes branch migration of Holliday junctions, and ino80 mutants are hypersensitive to DNA-damaging agents [57•]. These findings support the idea that common strategies and factors are shared by the different DNA transaction pathways to access DNA within chromatin, and raise the expectation that further connections will be found.
Conclusions Here we have discussed our current understanding of how nucleosomes are assembled de novo onto replicated or repaired DNA by using the activities of several histone chaperones and the incorporation of specifically modified histones, and through the coordination of PCNA. However, we still need to understand more precisely how these different chaperones function together, and how they contribute individually within specialized regions of the genome. It will also be of great interest to understand how their function can be regulated within the cell cycle. First insights may come from signalling pathways using phosphorylation [9,22,58], but additional controls including timing of expression and nuclear localization should be investigated. Although we have focused our attention on chromatin organization at the level of nucleosomes, chromatin is folded into higher order structures that give rise to functional nuclear domains. Importantly, variation introduced at the nucleosomal level can produce effects on these higher orders of chromatin organization. For example, the histone variant H2AX appears to be involved in nucleation of foci containing repair factors at sites of double-strand breaks [59], and the H3 variant CENP-A is found specifically at centromeres [60]. Furthermore, a dynamic interplay between methylation and phosphorylation of H3 is important for nucleation of chromatin folding [61•]. How these variations are introduced into chromatin and then maintained during replication and repair events will be crucial in evaluating which parameters are critical for functional genome integrity. A fundamental knowledge at these levels should have profound implications for our understanding of the etiology of several developmental disorders and of the genomic instability associated with cancer cells.
Acknowledgements We thank members of our group for critically reading this manuscript. J Mello was supported by the Institut Curie and a Chateaubriand Postdoctoral Fellowship. The ‘Chromatin dynamics’ team was supported by the Labellisation Ligue Nationale Contre le Cancer, the CNRS and the Institut Curie.
References and recommended reading Supporting this notion, CHRAC can facilitate efficient initiation of replication from the SV40 replication origin on a reconstituted chromatin template in vitro [55]. The repair factor cockayne syndrome B, which is required for the coupling of nucleotide excision repair to transcription, is part of the SWI2/SNF2 family and can remodel nucleosomes in vitro [56•]. Moreover, the remodeling complex INO80,
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Philpott A, Krude T, Laskey RA: Nuclear chaperones. Semin Cell Dev Biol 2000, 11:7-14.
2.
Verreault A: De novo nucleosome assembly: new pieces in an old puzzle. Genes Dev 2000, 14:1430-1438.
140
Chromosomes and expression mechanisms
3.
Ridgway P, Almouzni G: CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repair. J Cell Sci 2000, 113:2647-2658.
4.
Smith S, Stillman B: Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell 1989, 58:15-25.
5.
6.
7.
Verreault A, Kaufman PD, Kobayashi R, Stillman B: Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4. Cell 1996, 87:95-104. Gaillard PH, Martini EM, Kaufman PD, Stillman B, Moustacchi E, Almouzni G: Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I. Cell 1996, 86:887-896. Krude T: Chromatin assembly factor 1 (CAF-1) colocalizes with replication foci in HeLa cell nuclei. Exp Cell Res 1995, 220:304-311.
8. •
Taddei A, Roche D, Sibarita JB, Turner BM, Almouzni G: Duplication and maintenance of heterochromatin domains. J Cell Biol 1999, 147:1153-1166. This study reveals a specific importance of the incorporation of replicationassociated acetylated H4 isoforms into heterochromatin and their subsequent deacetylation, defining a unique time window for dynamic acetylation in these regions. 9.
Martini E, Roche DM, Marheineke K, Verreault A, Almouzni G: Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells. J Cell Biol 1998, 143:563-575.
10. Kaufman PD, Kobayashi R, Stillman B: Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I. Genes Dev 1997, 11:345-357. 11. Game JC, Kaufman PD: Role of Saccharomyces cerevisiae • chromatin assembly factor-I in repair of ultraviolet radiation damage in vivo. Genetics 1999, 151:485-497. Genetic analysis of mutants for CAF-1 combined with various rad mutants and UV sensitivity suggests that CAF-1 may have a role in the error-free post-replicative repair pathway. 12. Enomoto S, McCune-Zierath PD, Gerami-Nejad M, Sanders MA, Berman J: RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo. Genes Dev 1997, 11:358-370. 13. Enomoto S, Berman J: Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci. Genes Dev 1998, 12:219-232. 14. Monson EK, de Bruin D, Zakian VA: The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres. Proc Natl Acad Sci USA 1997, 94:13081-13086. 15. Murzina N, Verreault A, Laue E, Stillman B: Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol Cell 1999, 4:529-540. 16. Munakata T, Adachi N, Yokoyama N, Kuzuhara T, Horikoshi M: A human homologue of yeast anti-silencing factor has histone chaperone activity. Genes to Cells 2000, 5:221-233. 17. ••
Tyler JK, Adams CR, Chen SR, Kobayashi R, Kamakaka RT, Kadonaga JT: The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 1999, 402:555-560. From Drosophila embryo extracts, the authors describe the identification and purification of a novel assembly factor RCAF, which contains the homologue of budding yeast ASF1 together with specifically acetylated forms of H3 and H4. The purified complex can synergize with CAF-1 to assembly nucleosomes specifically on replicated DNA. In S. cerevisiae, genetic analysis indicates that ASF1 is essential for normal cell-cycle progression and may play a role in repair of double-strand breaks. 18. Le S, Davis C, Konopka JB, Sternglanz R: Two new S-phase-specific genes from Saccharomyces cerevisiae. Yeast 1997, 13:1029-1042. 19. Singer MS, Kahana A, Wolf AJ, Meisinger LL, Peterson SE, Goggin C, Mahowald M, Gottschling DE: Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae. Genetics 1998, 150:613-632. 20. Chang L, Loranger SS, Mizzen C, Ernst SG, Allis CD, Annunziato AT: Histones in transit: cytosolic histone complexes and diacetylation of H4 during nucleosome assembly in human cells. Biochem 1997, 36:469-480.
21. Ito T, Bulger M, Kobayashi R, Kadonaga JT: Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays. Mol Cell Biol 1996, 16:3112-3124. 22. Rodriguez P, Pelletier J, Price GB, Zannis-Hadjopoulos M: NAP-2: histone chaperone function and phosphorylation state through the cell cycle. J Mol Biol 2000, 298:225-238. 23. Ishimi Y, Kikuchi A: Identification and molecular cloning of yeast homolog of nucleosome assembly protein I which facilitates nucleosome assembly in vitro. J Biol Chem 1991, 266:7025-7029. 24. Ito T, Bulger M, Pazin MJ, Kobayashi R, Kadonaga JT: ACF, an ISWIcontaining and ATP-utilizing chromatin assembly and remodeling factor. Cell 1997, 90:145-155. 25. Ito T, Levenstein ME, Fyodorov DV, Kutach AK, Kobayashi R, • Kadonaga JT: ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev 1999, 13:1529-1539. The authors describe the identification of ACF from Drosophila, comprising Acf1 and ISWI subunits, which together function synergistically to catalyze the assembly of periodic nucleosomal arrays in the presence of NAP-1. 26. Zhang Z, Shibahara K, Stillman B: PCNA connects DNA replication • to epigenetic inheritance in yeast. Nature 2000, 408:221-225. In S. cerevisiae, mutations in PCNA are identified that lead to defects in silencing at telomeric and mating-type loci, and all of these mutants are also defective in the binding of the p150 subunit of CAF-1 to chromatin. PCNA CAF-1 double mutants suggest that PCNA effects epigenetic inheritance in both a CAF-1-dependent and -independent manner. 27. •
Rogner UC, Spyropoulos DD, Le Novere N, Changeux JP, Avner P: Control of neurulation by the nucleosome assembly protein-1-like 2. Nat Genet 2000, 25:431-435. Inactivation of the Nap1l2 gene in mice is found to be lethal, and a series of experiments suggest that mutation of Nap1l2 affects proliferation of neuronal precursor cells both in vivo and in vitro. 28. Shibahara K, Stillman B: Replication-dependent marking of DNA by •• PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 1999, 96:575-585. The authors find that nucleosome assembly can be temporally dissociated from replication in vitro, and using this strategy show that replicated DNA is marked by the presence of PCNA for subsequent CAF-1-dependent assembly. Furthermore, PCNA binds directly to CAF-1 p150 by co-immunoprecipitation, and the proteins colocalize at replication foci. 29. Moggs JG, Grandi P, Quivy JP, Jonsson ZO, Hubscher U, Becker PB, •• Almouzni G: A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage. Mol Cell Biol 2000, 20:1206-1218. The authors show that PCNA interacts directly with CAF-1 p150 by gluthathione S-transferase pull-down experiments, and they map critical regions on both proteins. Nucleosome assembly is triggered by the presence of single-strand breaks and gaps, and is dependent on PCNA; and both CAF-1 and PCNA are recruited to damaged DNA linked to beads. 30. Jonsson ZO, Hubscher U: Proliferating cell nuclear antigen: more than a clamp for DNA polymerases. Bioessays 1997, 19:967-975. 31. Warbrick E: The puzzle of PCNA’s many partners. Bioessays 2000, 22:997-1006. 32. Henderson DS, Banga SS, Grigliatti TA, Boyd JB: Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA. EMBO J 1994, 13:1450-1459. 33. Henderson DS, Wiegand UK, Norman DG, Glover DM: Mutual correction of faulty PCNA subunits in temperature-sensitive lethal mus209 mutants of Drosophila melanogaster. Genetics 2000, 154:1721-1733. 34. Thelen MP, Venclovas C, Fidelis K: A sliding clamp model for the Rad1 family of cell cycle checkpoint proteins. Cell 1999, 96:769-770. 35. Venclovas C, Thelen MP: Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamploading complexes. Nucleic Acids Res 2000, 28:2481-2493. 36. Sobel RE, Cook RG, Perry CA, Annunziato AT, Allis CD: Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4. Proc Natl Acad Sci USA 1995, 92:1237-1241.
The ins and outs of nucleosome assembly Mello and Almouzni
37.
Shibahara K, Verreault A, Stillman B: The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA in vitro. Proc Natl Acad Sci USA 2000, 97:7766-7771.
38. Kelly TJ, Qin S, Gottschling DE, Parthun MR: Type B histone acetyltransferase Hat1p participates in telomeric silencing. Mol Cell Biol 2000, 20:7051-7058. 39. Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL, Wolffe AP: • DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet 2000, 25:338-342. The mammalian DNA methyltransferase, DNMT1, is found to co-purify with HDAC1 and with the retinablastoma tumor suppressor gene product E2F1, and also to cooperate with the retinoplastoma protein in transcriptional repression, establishing a link between DNA methylation, histone deacetylation and sequence-specific DNA-binding activity. 40. Rountree MR, Bachman KE, Baylin SB: DNMT1 binds HDAC2 and a • new co-repressor, DMAP1, to form a complex at replication foci. Nat Genet 2000, 25:269-277. The authors show that the methyltransferase DNMT1 binds to HDAC2 and to a new protein, DMAP1, that displays intrinsic transcription repressive activity. DNMT1 and DMAP1 are targeted to replication foci throughout S phase, whereas HDAC2 is recruited late in S phase. These data suggest that DNMT1 may facilitate deacetylation of histones in heterochromatin after replication. 41. Ekwall K, Olsson T, Turner BM, Cranston G, Allshire RC: Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres. Cell 1997, 91:1021-1032. 42. Grewal SI, Bonaduce MJ, Klar AJ: Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast. Genetics 1998, 150:563-576. 43. Taddei A, Maison C, Roche D, Almouzni G: Reversible disruption of • pericentric heterochromatin and centromere function by inhibiting deacetylases in mammalian cells. Nat Cell Biol 2001, 3:114-120. In mammalian cells, maintaining histone acetylation by treatment with a specific deacetylase inhibitor is shown to cause relocation of pericentric heterochromatin to the nuclear periphery, accompanied by a loss of HP1 at these regions and subsequent defects in chromosome segregation, indicating that underacetylation has a critical role in proper function of heterochromatin regions. 44. LeRoy G, Loyola A, Lane WS, Reinberg D: Purification and characterization of a human factor that assembles and remodels chromatin. J Biol Chem 2000, 275:14787-14790. 45. Guschin D, Geiman TM, Kikyo N, Tremethick DJ, Wolffe AP, Wade PA: Multiple ISWI ATPase complexes from xenopus laevis. Functional conservation of an ACF/CHRAC homolog. J Biol Chem 2000, 275:35248-35255 46. Varga-Weisz PD, Wilm M, Bonte E, Dumas K, Mann M, Becker PB: Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature 1997, 388:598-602. 47.
Poot RA, Dellaire G, Hulsmann BB, Grimaldi MA, Corona DF, Becker PB, Bickmore WA, Varga-Weisz PD: HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins. EMBO J 2000, 19:3377-3387.
48. LeRoy G, Orphanides G, Lane WS, Reinberg D: Requirement of RSF and FACT for transcription of chromatin templates in vitro. Science 1998, 282:1900-1904.
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49. Peterson CL, Workman JL: Promoter targeting and chromatin remodeling by the SWI/SNF complex. Curr Opin Genet Dev 2000, 10:187-192. 50. Guschin D, Wolffe AP: SWItched-on mobility. Curr Biol 1999, 9:R742-R746. 51. Aalfs JD, Kingston RE: What does ‘chromatin remodeling’ mean? Trends Biochem Sci 2000, 25:548-555. 52. Moggs JG, Almouzni G: Chromatin rearrangements during nucleotide excision repair. Biochimie 1999, 81:45-52. 53. Meijer M, Smerdon MJ: Accessing DNA damage in chromatin: insights from transcription. Bioessays 1999, 21:596-603. 54. Thoma F: Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair. EMBO J 1999, 18:6585-6598. 55. Alexiadis V, Varga-Weisz PD, Bonte E, Becker PB, Gruss C: In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication. EMBO J 1998, 17:3428-3438. 56. Citterio E, Van Den Boom V, Schnitzler G, Kanaar R, Bonte E, • Kingston RE, Hoeijmakers JH, Vermeulen W: ATP-Dependent chromatin remodeling by the cockayne syndrome B DNA repair-transcription-coupling factor. Mol Cell Biol 2000, 20:7643-7653. This study shows for the first time that a known repair protein, the transcription-coupled repair factor CSB, can remodel chromatin structure and interact directly with core histones. 57. •
Shen X, Mizuguchi G, Hamiche A, Wu C: A chromatin remodelling complex involved in transcription and DNA processing. Nature 2000, 406:541-544. The authors describe the purification from of an S. cerevisiae 12-subunit complex, INO80, containing an ATPase and two proteins related to the bacterial RuvB DNA helicase. They present data indicating that chromatin remodeling by this complex may have a role both in transcription and DNA repair. 58. Keller C, Krude T: Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis. J Biol Chem 2000, 275:35512-35521. 59. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM: A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 2000, 10:886-895. 60. Shelby RD, Monier K, Sullivan KF: Chromatin assembly at kinetochores Is uncoupled from DNA replication. J Cell Biol 2000, 151:1113-1118 61. Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schmid M, • Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 2000, 406:593-599. The authors show that human SUV39H1 and its murine homologue encode histone H3-specific methyltransferases that methylate lysine 9 of H3 in vitro. Deregulation of the enzyme in vivo modulates phosphorylation of H3 serine 10 and induces aberrant mitotic divisions, suggesting a functional interdependence of H3 tail modifications in regulating higher order chromatin.
The ins and outs of nucleosome assembly Jill A Mello* and Geneviève Almouzni† De novo nucleosome assembly coupled to DNA replication and repair in vitro involves the histone chaperone chromatin assembly factor 1 (CAF-1). Recent studies support a model in which CAF-1 can be targeted to newly synthesized DNA through a direct interaction with proliferating cell nuclear antigen (PCNA) and can act synergistically with a newly identified histone chaperone. Insights have also been obtained into mechanisms by which this CAF-1-dependent pathway can establish a repressed chromatin state. Addresses Institut Curie, Research section, UMR 218 du Centre National de la Recherche Scientifique (CNRS), 75248 Paris cedex 05, France *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Genetics & Development 2001, 11:136–141 0959-437X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ACF ATP-utilizing chromatin assembly and remodeling factor ASF1 anti-silencing factor 1 CAF-1 chromatin assembly factor 1 CHRAC chromatin accessibility complex HP1 heterochromatin-binding protein 1 ISWI SWI2-related imitation switch NAP-1 nucleosome assembly protein 1 PCNA proliferating cell nuclear antigen
Introduction Chromatin plays a critical role in the regulation of DNA metabolic processes, including replication, repair and gene expression. Efficient assembly of nucleosomes — the fundamental repeating units of chromatin — onto newly synthesized DNA is thus essential for maintaining proper genome function. At the replication fork, parental nucleosomes are redistributed between the two nascent chromatids, and de novo nucleosome assembly provides the required nucleosome complement. Evidence indicates that a de novo pathway also operates during the repair of DNA damage. Here we discuss recent insights into the mechanism of de novo nucleosome assembly and how the requisite proteins may participate to impart a transcriptional state.
Nucleosome assembly and histone chaperones: a coordinated network? Nucleosome assembly follows a two-step process in which a tetramer of histones H3 and H4 is first deposited onto DNA, and subsequently two H2A–H2B heterodimers are deposited to yield an octamer core around which 146 base pairs (bp) of DNA is wrapped. The ordered assembly of nucleosomes within the nucleus is thought to be mediated by histone chaperones [1,2] — proteins that bind and effectively neutralize highly charged histones, thereby preventing their nonspecific aggregation with DNA. Several histone chaperones have been identified by using in vitro
reconstitution experiments, and a current challenge is to understand how they function — either alone or in a coordinated manner — in the assembly of nucleosomes. The best-characterized histone chaperone is chromatin assembly factor 1 (CAF-1) — a multisubunit protein, which in humans contains the three subunits p150, p60 and p48, whose function is evolutionarily conserved [3]. CAF-1 is associated with newly synthesized forms of H3 and H4. The most striking feature of CAF-1 is its unique ability to promote preferentially nucleosome assembly onto newly synthesized DNA, either during replication [4,5] or during nucleotide excision repair [6] in vitro; this makes CAF-1 a strong candidate for a coordinator of nucleosome assembly during DNA synthesis in vivo. The localization of CAF-1 to replication foci during S phase, at both euchromatin and heterochromatin regions [7,8•], and its recruitment to chromatin in G1- or G2-phase cells that have undergone UV DNA damage [9] strongly support this idea. Null mutants of CAF-1 homologues in Saccharomyces cerevisiae are mildly sensitive to UV radiation but are otherwise perfectly viable [10,11•], suggesting that alternate de novo assembly pathways are operative, at least during S phase, in this organism. Mutants of CAF-1 in budding yeast also show defects in transcriptional silencing at telomeres and at the mating-type loci HMLα and HMRa [10,12–14], indicating that CAF-1 is involved in maintaining heterochromatin at these loci. In mammalian cells, an interaction between CAF-1 and heterochromatin-binding protein 1 (HP1) may be of possible relevance [15]. To clarify further the general role of CAF-1 in nucleosome assembly coordinated to DNA synthesis, both during and outside S phase, it will be critical to assess its in vivo significance in higher eukaryotes. It will also be important to understand why certain chromatin regions are particularly stringent in their requirement for CAF-1. The apparent existence in budding yeast of redundant de novo assembly pathways has spurred the search for new assembly factors. One outcome is the recent purification of a new nucleosome assembly complex, RCAF, from Drosophila embryo extracts; RCAF was identified by its ability to synergize with CAF-1 to promote nucleosome assembly during DNA replication in vitro. RCAF comprises the highly evolutionarily conserved protein anti-silencing factor-1 (ASF1), of which the human homologue displays histone chaperone activity [16], together with newly synthesized forms of H3 and H4 [17••]. Mutants of Asf1, either alone or in combination with mutants of CAF-1, are viable in yeast, which indicates that other assembly pathways exist. When compared with CAF-1, however, Asf1 mutants display additional growth defects and
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Figure 1 A model for targeting nucleosome assembly to DNA transactions. De novo nucleosome assembly involves the deposition of a tetramer of newly synthesized forms of histones H3 and H4 in a process involving the histone chaperone CAF-1, followed by the addition of two H2A–H2B heterodimers. The specificity for targeting nucleosome assembly to DNA transactions is achieved through a direct interaction of CAF-1 with PCNA, either at sites of DNA repair (left) or at the replication fork (right). In addition, we present the hypothesis that another histone chaperone, ASF1, may deliver newly synthesized H3 and H4 to DNA targeted through CAF-1, either by: (i) shuttling the histones to CAF-1 prior to its interaction with PCNA or (ii) directly at sites of replication or repair. Alternatively, ASF1 may deliver H3 and H4 in a distinct, CAF-1 independent manner.
(H3–H4)2 ASF1 (i) (ii)
2 X H2A–H2B
(ii) (H3–H4)2 CAF-1 CAF-1
CAF-1
PCNA PCNA DNA repair site
exhibit sensitivity to a broader range of DNA-damaging agents [17••,18]. Furthermore, whereas a defect in Asf1 shows very little effect on silencing at telomeres or mating-type loci, a double mutant of CAF-1 and Asf1 displays more pronounced silencing defects than does either single mutant [17••,18,19]. From these studies, we can infer that Asf1 and CAF-1 are to some degree functionally distinct, and that both participate in heterochromatic silencing. Further clues to the role of Asf1 are awaited eagerly. We are tempted to speculate, however, that Asf1 may act as a histone donor for CAF-1, shuttling H3–H4 to CAF-1 that is localized at sites of assembly. Indeed, the general idea that chaperones might function within a coordinated assembly line to deliver histones to target DNA is an attractive strategy for achieving a fast, efficient process. Moreover, introducing specialized chaperones, which could perhaps be associated with specifically modified histones or histone variants, into a chaperone network might be a way to achieve temporal or regional control for establishing specialized chromatin structures. If we consider that nucleosome assembly is achieved through the function of a coordinated network, it is likely that the deposition of H2A–H2B dimers on the H3–H4 tetramer is also mediated by a histone chaperone. Nucleosome assembly protein 1 (NAP-1) is an evolutionarily conserved histone chaperone that is associated with H2A–H2B both in human cells [20] and in Drosophila embryo extracts [21]. Both Drosophila NAP-1 and the highly related human protein NAP-2 [22] undergo rapid localization to the nucleus in S phase, suggesting that they have a role in DNA replication. But, in a manner independent of replication, NAP-1 can promote nucleosome assembly in the presence of all core histones [23] and can cooperate with ATP-utilizing chromatin assembly and remodeling factor (ACF) to assemble regularly spaced
Replication fork Current Opinion in Genetics & Development
nucleosomal arrays [24,25•]. Thus if NAP-1 does play a role in de novo nucleosome assembly, it may rely on the initial specificity of CAF-1 or other chaperones for newly synthesized DNA in the first step of the process. In this regard, the recently reported ability of CAF-1 p60 to interact with H2A and H2B [26•] presents the possibility that NAP-1 or other factors might deliver these histones to CAF-1 localized at sites of DNA synthesis, which might, in turn, facilitate their deposition through p60. In addition to the idea that chaperones might cooperate as part of a network, it is important to stress the non-mutually exclusive possibility that some chaperones might possess a dedicated function. The histone chaperones nucleoplasmin-1 and N1/N2 are associated with H2A–H2B and H3–H4, respectively [1]. To date, these proteins have been identified only in oocytes of Xenopus and Drosophila, and may function exclusively during the rapid cell divisions required during early embryogenesis. Interestingly, a recent study in mice of the X-linked NAP-1 gene family member, Nap1l2, has shown that its encoded protein is cytoplasmic in G1 but localizes to chromatin during S phase, and is expressed mainly in neurons [27•]. In addition, inactivation of Nap1l2 was lethal from mid-gestation, but caused severe defects in neuronal cell proliferation in surviving mutant chimeric embryos [27•]. These results suggest that Nap1/2 may be a tissue-specific chromatin factor. Thus, it is reasonable to speculate that other tissue-specific chaperones exist and may have dedicated roles during development.
Targeting nucleosome assembly to DNA transactions Just as we have begun to understand how molecular chaperones mediate the deposition of histones onto newly synthesized DNA, light has also been recently shed on another critical question: how are these chaperones brought to the sites of DNA transactions? The remarkable
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specificity of CAF-1 for nucleosome assembly on replicated and repaired DNA in vitro has been explained recently by its ability to recognize DNA that is topologically marked by the presence of the proliferating cell nuclear antigen (PCNA) [28••,29••]. PCNA is a homotrimeric protein that forms a sliding clamp around DNA and functions as a DNA polymerase processivity factor during replication and nucleotide excision repair [30]. In vivo, co-immunolocalization of PCNA and CAF-1 at replication foci and to chromatin of UV-irradiated cells strongly supports a role for PCNA as a recruitment factor for chromatin assembly machinery [9,28••]. At a biochemical level, the human CAF-1 p150 subunit interacts directly with PCNA through contacts with the interdomain connecting loop on the outer, front side of the PCNA ring — a region known to interact with several other proteins [26•,29••]. Indeed, PCNA interacts with many proteins [31], and a picture is beginning to emerge in which PCNA acts as a central coordinator for replication, repair, epigenetic inheritance and cell-cycle control. A more detailed description of how p150 and other interactor proteins compete for binding to PCNA, or alternatively are accommodated by the homotrimeric structure, would help to elucidate how several activities are coordinated at the replication fork and at repair sites. A PCNA-dependent chromatin assembly pathway targeting CAF-1 to DNA regions may have profound functional consequences, both in replication and repair. A recent study of PCNA mutants in S. cerevisiae has shown that, reminiscent of defects in CAF-1, several mutations in PCNA significantly decrease silencing at telomeres and at the mating-type HMR locus [26•]. This finding is consistent with the identified of a PCNA mutant in Drosophila which suppresses repression in the vicinity of heterochromatin [32,33]. All PCNA mutants that reduced silencing were also defective in the stable binding of the CAF-1 p150 subunit to chromatin, reinforcing the importance of their physical interaction in establishing specialized chromatin states. Interestingly, several PCNA mutations in combination with CAF-1 mutations displayed a synergistic decrease in silencing, suggesting that PCNA may also affect silencing in a manner at least partially independent of CAF-1, perhaps through the recruitment of other histone chaperones [26•]. In the context of replication, Shibahara and Stillman [28••] have proposed that asymmetric loading of PCNA on the leading and lagging strands at the replication fork may provide the opportunity to establish different chromatin structures on the two sister chromatids, thereby providing a mechanism for epigenetic inheritance. In the context of DNA repair, the specific chromatin structure established through this PCNA-dependent mechanism is, at this point in time, an open issue. It is possible that the PCNA-dependent targeting of chromatin assembly to DNA repair may set up a repressive chromatin structure, shutting down
DNA metabolism in the region until repair is complete, perhaps as part of a coordinated cell-cycle response to DNA damage [29••]. Compellingly, the repair and cellcycle checkpoint proteins Rad1/Rad9/Hus1 are predicted to form a PCNA-like heterotrimeric ring [34,35]. The possibility that CAF-1-dependent nucleosome assembly could be connected to this checkpoint pathway through a direct interaction between this complex and CAF-1 p150 deserves future investigation.
A cycle of histone acetylation and deacetylation and nucleosome assembly Newly synthesized histones H3 and H4 undergo a transient acetylation at their amino-terminal tails during S phase, before deposition onto replicated DNA, and are soon after deacetylated. In particular, newly synthesized H4 exhibits a highly conserved acetylation pattern at lysines 5 and 12 [36], and, importantly, this post-translational modification is distinct from the acetylation of chromatin-deposited histones associated with gene expression in euchromatin. The functional significance of post-translational H3–H4 acetylation for nucleosome assembly remains unknown, although it is clearly not required for core particle formation by dialysis in vitro. Beyond this, it has been suggested that the acetylation may be needed for nuclear import of histones or for their deposition by chromatin assembly factors. Addressing the latter possibility, a recent study has shown that CAF-1 can bind stably to tetramers of H3–H4 lacking their amino-terminal tails, and, moreover, can efficiently promote their preferential loading onto replicated DNA in vitro [37]. It nevertheless remains possible that acetylation is important for interaction with other histone chaperones, such as Asf1. An alternative role for H3–H4 acetylation might be in events that occur after deposition, either facilitating further chromatin maturation steps, or acting as a transient ‘marking’ mechanism for monitoring cell-cycle progression. In any case, it is noteworthy that in budding yeast, the type B histone acetyltransferase that modifies newly synthesized H4 is important for telomeric silencing [38], indicating functional significance for the acetylation of newly synthesized histones, at least in these regions. The deacetylation event that follows replication, like that of acetylation, is poorly understood. Rapid progress may come with the recent identification of several strong candidate enzymes for the deacetylation reaction [39•,40•]. In the genome, heterochromatin in particular exists in an underacetylated state. Of relevance, studies in Schizosaccharomyces pombe [41,42], and more recently in mammalian cells [43•], have shown that inhibition of deacetylation leads to delocalization of HP1, as well as to severe defects in centromeric heterochromatin regions and chromosome segregation. Thus, histone deacetylation seems to be critical for heterochromatin regions and for maintaining genomic integrity.
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At this point, it is worth recalling that in budding yeast the phenotype associated with mutants of CAF-1 is also revealed at heterochromatin regions. Interestingly, the smallest subunit of CAF-1, p48, associates with histone H4 and also with the catalytic subunit of the human deacetylase HD1 [3], suggesting a mechanism by which deacetylation might be tightly coupled to the assembly process. The fact that an observable phenotype associated with defects in many steps of the de novo nucleosome assembly pathway manifests primarily at heterochromatin regions may be understandable if we consider that within heterochromatin, deposition of acetylated H3–H4 and subsequent deacetylation appears to occur only one time during S phase [8•], whereas an overlapping dynamic acetylation/deacetylation process operates continuously in euchromatin. This situation would render heterochromatin more sensitive to changes introduced to the system.
Remodeling factors and nucleosome mobility Nucleosome assembly by histone chaperones alone is insufficient to obtain the long, regularly spaced nucleosomal arrays found in eukaryotic chromosomes, and requires the additional function of ATP-utilizing factors. Several factors have been purified from human cell, Drosophila embryo and Xenopus egg extracts on the basis of their ability to promote ATP-dependent nucleosome spacing, including ACF [24,44,45], the chromatin accessibility complex (CHRAC) [46,47], and the remodeling and spacing factor [48]. All remodeling factors identified to date contain a nucleosome-dependent ATP-utilizing subunit, many belonging to either the SWI/SNF or SWI2-related imitation switch (ISWI) chromatin remodelling families [49,50]. This ATPase subunit is thought to promote nucleosome spacing by facilitating nucleosome mobility, perhaps through a partial disruption of the nucleosome structure. The precise mechanism by which exact intranucleosomal spacing is determined, however, remains unclear. The ability of remodeling factors to disrupt histone–DNA interactions has been implicated clearly in the ‘opening up’ of the chromatin structure that occurs during transcription [51]. The extent to which nucleosomes are disassembled or remodeled at replication origins and sites of DNA repair remains to be determined, although it is clear that changes in chromatin structure occur [52]. Thus, it is equally important to learn how specific DNA regions within the chromatin architecture are made accessible to the replication or repair machinery, and it is conceivable that the ATP-dependent remodeling activities used in transcription may also be used in these contexts [53,54].
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which contains an ISWI-like ATPase, also contains two proteins related to the bacterial RuvB DNA helicase that catalyzes branch migration of Holliday junctions, and ino80 mutants are hypersensitive to DNA-damaging agents [57•]. These findings support the idea that common strategies and factors are shared by the different DNA transaction pathways to access DNA within chromatin, and raise the expectation that further connections will be found.
Conclusions Here we have discussed our current understanding of how nucleosomes are assembled de novo onto replicated or repaired DNA by using the activities of several histone chaperones and the incorporation of specifically modified histones, and through the coordination of PCNA. However, we still need to understand more precisely how these different chaperones function together, and how they contribute individually within specialized regions of the genome. It will also be of great interest to understand how their function can be regulated within the cell cycle. First insights may come from signalling pathways using phosphorylation [9,22,58], but additional controls including timing of expression and nuclear localization should be investigated. Although we have focused our attention on chromatin organization at the level of nucleosomes, chromatin is folded into higher order structures that give rise to functional nuclear domains. Importantly, variation introduced at the nucleosomal level can produce effects on these higher orders of chromatin organization. For example, the histone variant H2AX appears to be involved in nucleation of foci containing repair factors at sites of double-strand breaks [59], and the H3 variant CENP-A is found specifically at centromeres [60]. Furthermore, a dynamic interplay between methylation and phosphorylation of H3 is important for nucleation of chromatin folding [61•]. How these variations are introduced into chromatin and then maintained during replication and repair events will be crucial in evaluating which parameters are critical for functional genome integrity. A fundamental knowledge at these levels should have profound implications for our understanding of the etiology of several developmental disorders and of the genomic instability associated with cancer cells.
Acknowledgements We thank members of our group for critically reading this manuscript. J Mello was supported by the Institut Curie and a Chateaubriand Postdoctoral Fellowship. The ‘Chromatin dynamics’ team was supported by the Labellisation Ligue Nationale Contre le Cancer, the CNRS and the Institut Curie.
References and recommended reading Supporting this notion, CHRAC can facilitate efficient initiation of replication from the SV40 replication origin on a reconstituted chromatin template in vitro [55]. The repair factor cockayne syndrome B, which is required for the coupling of nucleotide excision repair to transcription, is part of the SWI2/SNF2 family and can remodel nucleosomes in vitro [56•]. Moreover, the remodeling complex INO80,
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Philpott A, Krude T, Laskey RA: Nuclear chaperones. Semin Cell Dev Biol 2000, 11:7-14.
2.
Verreault A: De novo nucleosome assembly: new pieces in an old puzzle. Genes Dev 2000, 14:1430-1438.
140
Chromosomes and expression mechanisms
3.
Ridgway P, Almouzni G: CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repair. J Cell Sci 2000, 113:2647-2658.
4.
Smith S, Stillman B: Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell 1989, 58:15-25.
5.
6.
7.
Verreault A, Kaufman PD, Kobayashi R, Stillman B: Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4. Cell 1996, 87:95-104. Gaillard PH, Martini EM, Kaufman PD, Stillman B, Moustacchi E, Almouzni G: Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I. Cell 1996, 86:887-896. Krude T: Chromatin assembly factor 1 (CAF-1) colocalizes with replication foci in HeLa cell nuclei. Exp Cell Res 1995, 220:304-311.
8. •
Taddei A, Roche D, Sibarita JB, Turner BM, Almouzni G: Duplication and maintenance of heterochromatin domains. J Cell Biol 1999, 147:1153-1166. This study reveals a specific importance of the incorporation of replicationassociated acetylated H4 isoforms into heterochromatin and their subsequent deacetylation, defining a unique time window for dynamic acetylation in these regions. 9.
Martini E, Roche DM, Marheineke K, Verreault A, Almouzni G: Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells. J Cell Biol 1998, 143:563-575.
10. Kaufman PD, Kobayashi R, Stillman B: Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I. Genes Dev 1997, 11:345-357. 11. Game JC, Kaufman PD: Role of Saccharomyces cerevisiae • chromatin assembly factor-I in repair of ultraviolet radiation damage in vivo. Genetics 1999, 151:485-497. Genetic analysis of mutants for CAF-1 combined with various rad mutants and UV sensitivity suggests that CAF-1 may have a role in the error-free post-replicative repair pathway. 12. Enomoto S, McCune-Zierath PD, Gerami-Nejad M, Sanders MA, Berman J: RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo. Genes Dev 1997, 11:358-370. 13. Enomoto S, Berman J: Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci. Genes Dev 1998, 12:219-232. 14. Monson EK, de Bruin D, Zakian VA: The yeast Cac1 protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres. Proc Natl Acad Sci USA 1997, 94:13081-13086. 15. Murzina N, Verreault A, Laue E, Stillman B: Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol Cell 1999, 4:529-540. 16. Munakata T, Adachi N, Yokoyama N, Kuzuhara T, Horikoshi M: A human homologue of yeast anti-silencing factor has histone chaperone activity. Genes to Cells 2000, 5:221-233. 17. ••
Tyler JK, Adams CR, Chen SR, Kobayashi R, Kamakaka RT, Kadonaga JT: The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 1999, 402:555-560. From Drosophila embryo extracts, the authors describe the identification and purification of a novel assembly factor RCAF, which contains the homologue of budding yeast ASF1 together with specifically acetylated forms of H3 and H4. The purified complex can synergize with CAF-1 to assembly nucleosomes specifically on replicated DNA. In S. cerevisiae, genetic analysis indicates that ASF1 is essential for normal cell-cycle progression and may play a role in repair of double-strand breaks. 18. Le S, Davis C, Konopka JB, Sternglanz R: Two new S-phase-specific genes from Saccharomyces cerevisiae. Yeast 1997, 13:1029-1042. 19. Singer MS, Kahana A, Wolf AJ, Meisinger LL, Peterson SE, Goggin C, Mahowald M, Gottschling DE: Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae. Genetics 1998, 150:613-632. 20. Chang L, Loranger SS, Mizzen C, Ernst SG, Allis CD, Annunziato AT: Histones in transit: cytosolic histone complexes and diacetylation of H4 during nucleosome assembly in human cells. Biochem 1997, 36:469-480.
21. Ito T, Bulger M, Kobayashi R, Kadonaga JT: Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays. Mol Cell Biol 1996, 16:3112-3124. 22. Rodriguez P, Pelletier J, Price GB, Zannis-Hadjopoulos M: NAP-2: histone chaperone function and phosphorylation state through the cell cycle. J Mol Biol 2000, 298:225-238. 23. Ishimi Y, Kikuchi A: Identification and molecular cloning of yeast homolog of nucleosome assembly protein I which facilitates nucleosome assembly in vitro. J Biol Chem 1991, 266:7025-7029. 24. Ito T, Bulger M, Pazin MJ, Kobayashi R, Kadonaga JT: ACF, an ISWIcontaining and ATP-utilizing chromatin assembly and remodeling factor. Cell 1997, 90:145-155. 25. Ito T, Levenstein ME, Fyodorov DV, Kutach AK, Kobayashi R, • Kadonaga JT: ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev 1999, 13:1529-1539. The authors describe the identification of ACF from Drosophila, comprising Acf1 and ISWI subunits, which together function synergistically to catalyze the assembly of periodic nucleosomal arrays in the presence of NAP-1. 26. Zhang Z, Shibahara K, Stillman B: PCNA connects DNA replication • to epigenetic inheritance in yeast. Nature 2000, 408:221-225. In S. cerevisiae, mutations in PCNA are identified that lead to defects in silencing at telomeric and mating-type loci, and all of these mutants are also defective in the binding of the p150 subunit of CAF-1 to chromatin. PCNA CAF-1 double mutants suggest that PCNA effects epigenetic inheritance in both a CAF-1-dependent and -independent manner. 27. •
Rogner UC, Spyropoulos DD, Le Novere N, Changeux JP, Avner P: Control of neurulation by the nucleosome assembly protein-1-like 2. Nat Genet 2000, 25:431-435. Inactivation of the Nap1l2 gene in mice is found to be lethal, and a series of experiments suggest that mutation of Nap1l2 affects proliferation of neuronal precursor cells both in vivo and in vitro. 28. Shibahara K, Stillman B: Replication-dependent marking of DNA by •• PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 1999, 96:575-585. The authors find that nucleosome assembly can be temporally dissociated from replication in vitro, and using this strategy show that replicated DNA is marked by the presence of PCNA for subsequent CAF-1-dependent assembly. Furthermore, PCNA binds directly to CAF-1 p150 by co-immunoprecipitation, and the proteins colocalize at replication foci. 29. Moggs JG, Grandi P, Quivy JP, Jonsson ZO, Hubscher U, Becker PB, •• Almouzni G: A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage. Mol Cell Biol 2000, 20:1206-1218. The authors show that PCNA interacts directly with CAF-1 p150 by gluthathione S-transferase pull-down experiments, and they map critical regions on both proteins. Nucleosome assembly is triggered by the presence of single-strand breaks and gaps, and is dependent on PCNA; and both CAF-1 and PCNA are recruited to damaged DNA linked to beads. 30. Jonsson ZO, Hubscher U: Proliferating cell nuclear antigen: more than a clamp for DNA polymerases. Bioessays 1997, 19:967-975. 31. Warbrick E: The puzzle of PCNA’s many partners. Bioessays 2000, 22:997-1006. 32. Henderson DS, Banga SS, Grigliatti TA, Boyd JB: Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA. EMBO J 1994, 13:1450-1459. 33. Henderson DS, Wiegand UK, Norman DG, Glover DM: Mutual correction of faulty PCNA subunits in temperature-sensitive lethal mus209 mutants of Drosophila melanogaster. Genetics 2000, 154:1721-1733. 34. Thelen MP, Venclovas C, Fidelis K: A sliding clamp model for the Rad1 family of cell cycle checkpoint proteins. Cell 1999, 96:769-770. 35. Venclovas C, Thelen MP: Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamploading complexes. Nucleic Acids Res 2000, 28:2481-2493. 36. Sobel RE, Cook RG, Perry CA, Annunziato AT, Allis CD: Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4. Proc Natl Acad Sci USA 1995, 92:1237-1241.
The ins and outs of nucleosome assembly Mello and Almouzni
37.
Shibahara K, Verreault A, Stillman B: The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA in vitro. Proc Natl Acad Sci USA 2000, 97:7766-7771.
38. Kelly TJ, Qin S, Gottschling DE, Parthun MR: Type B histone acetyltransferase Hat1p participates in telomeric silencing. Mol Cell Biol 2000, 20:7051-7058. 39. Robertson KD, Ait-Si-Ali S, Yokochi T, Wade PA, Jones PL, Wolffe AP: • DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat Genet 2000, 25:338-342. The mammalian DNA methyltransferase, DNMT1, is found to co-purify with HDAC1 and with the retinablastoma tumor suppressor gene product E2F1, and also to cooperate with the retinoplastoma protein in transcriptional repression, establishing a link between DNA methylation, histone deacetylation and sequence-specific DNA-binding activity. 40. Rountree MR, Bachman KE, Baylin SB: DNMT1 binds HDAC2 and a • new co-repressor, DMAP1, to form a complex at replication foci. Nat Genet 2000, 25:269-277. The authors show that the methyltransferase DNMT1 binds to HDAC2 and to a new protein, DMAP1, that displays intrinsic transcription repressive activity. DNMT1 and DMAP1 are targeted to replication foci throughout S phase, whereas HDAC2 is recruited late in S phase. These data suggest that DNMT1 may facilitate deacetylation of histones in heterochromatin after replication. 41. Ekwall K, Olsson T, Turner BM, Cranston G, Allshire RC: Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres. Cell 1997, 91:1021-1032. 42. Grewal SI, Bonaduce MJ, Klar AJ: Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast. Genetics 1998, 150:563-576. 43. Taddei A, Maison C, Roche D, Almouzni G: Reversible disruption of • pericentric heterochromatin and centromere function by inhibiting deacetylases in mammalian cells. Nat Cell Biol 2001, 3:114-120. In mammalian cells, maintaining histone acetylation by treatment with a specific deacetylase inhibitor is shown to cause relocation of pericentric heterochromatin to the nuclear periphery, accompanied by a loss of HP1 at these regions and subsequent defects in chromosome segregation, indicating that underacetylation has a critical role in proper function of heterochromatin regions. 44. LeRoy G, Loyola A, Lane WS, Reinberg D: Purification and characterization of a human factor that assembles and remodels chromatin. J Biol Chem 2000, 275:14787-14790. 45. Guschin D, Geiman TM, Kikyo N, Tremethick DJ, Wolffe AP, Wade PA: Multiple ISWI ATPase complexes from xenopus laevis. Functional conservation of an ACF/CHRAC homolog. J Biol Chem 2000, 275:35248-35255 46. Varga-Weisz PD, Wilm M, Bonte E, Dumas K, Mann M, Becker PB: Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature 1997, 388:598-602. 47.
Poot RA, Dellaire G, Hulsmann BB, Grimaldi MA, Corona DF, Becker PB, Bickmore WA, Varga-Weisz PD: HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins. EMBO J 2000, 19:3377-3387.
48. LeRoy G, Orphanides G, Lane WS, Reinberg D: Requirement of RSF and FACT for transcription of chromatin templates in vitro. Science 1998, 282:1900-1904.
141
49. Peterson CL, Workman JL: Promoter targeting and chromatin remodeling by the SWI/SNF complex. Curr Opin Genet Dev 2000, 10:187-192. 50. Guschin D, Wolffe AP: SWItched-on mobility. Curr Biol 1999, 9:R742-R746. 51. Aalfs JD, Kingston RE: What does ‘chromatin remodeling’ mean? Trends Biochem Sci 2000, 25:548-555. 52. Moggs JG, Almouzni G: Chromatin rearrangements during nucleotide excision repair. Biochimie 1999, 81:45-52. 53. Meijer M, Smerdon MJ: Accessing DNA damage in chromatin: insights from transcription. Bioessays 1999, 21:596-603. 54. Thoma F: Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair. EMBO J 1999, 18:6585-6598. 55. Alexiadis V, Varga-Weisz PD, Bonte E, Becker PB, Gruss C: In vitro chromatin remodelling by chromatin accessibility complex (CHRAC) at the SV40 origin of DNA replication. EMBO J 1998, 17:3428-3438. 56. Citterio E, Van Den Boom V, Schnitzler G, Kanaar R, Bonte E, • Kingston RE, Hoeijmakers JH, Vermeulen W: ATP-Dependent chromatin remodeling by the cockayne syndrome B DNA repair-transcription-coupling factor. Mol Cell Biol 2000, 20:7643-7653. This study shows for the first time that a known repair protein, the transcription-coupled repair factor CSB, can remodel chromatin structure and interact directly with core histones. 57. •
Shen X, Mizuguchi G, Hamiche A, Wu C: A chromatin remodelling complex involved in transcription and DNA processing. Nature 2000, 406:541-544. The authors describe the purification from of an S. cerevisiae 12-subunit complex, INO80, containing an ATPase and two proteins related to the bacterial RuvB DNA helicase. They present data indicating that chromatin remodeling by this complex may have a role both in transcription and DNA repair. 58. Keller C, Krude T: Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis. J Biol Chem 2000, 275:35512-35521. 59. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM: A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 2000, 10:886-895. 60. Shelby RD, Monier K, Sullivan KF: Chromatin assembly at kinetochores Is uncoupled from DNA replication. J Cell Biol 2000, 151:1113-1118 61. Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schmid M, • Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 2000, 406:593-599. The authors show that human SUV39H1 and its murine homologue encode histone H3-specific methyltransferases that methylate lysine 9 of H3 in vitro. Deregulation of the enzyme in vivo modulates phosphorylation of H3 serine 10 and induces aberrant mitotic divisions, suggesting a functional interdependence of H3 tail modifications in regulating higher order chromatin.