- Email: [email protected]
Nucleosome assembly: the CAF and the HAT
369
Nucleosome assembly: the CAF and the HAT Paul D Kaufman Recent data argue strongly that a protein complex termed chromatin assembly factor-l (CAF-I) plays a major role in de nova nucleosome assembly during DNA replication. Human CAF-I deposits newly synthesized, acetylated histones onto replicated DNA in vitro and localizes to sites of DNA replication in S-phase cells. Specific lysines of the histones used for nucleosome assembly are acetylated; in the past year the first gene encoding a histone acetyltransferase was cloned. However, mechanistic links between histone acetylation and nucleosome assembly have not been established in vivo or in vitro.
two H2A-H2B heterodimers complete the nucleosome.
are added
subsequently
to
E-amino groups of lysines near the amino-terminal regions of histones undergo reversible acetylation (reviewed in [9,10]). This modification can occur during two distinct biological processes. First, histones already incorporated into nuclear chromatin can become acetylated. Second, a different pattern of acetylation is observed on histones that are newly synthesized in the cytoplasm. This review, which concerns assembly of newly synthesized histones into nucleosomes, will focus on the latter type of acetylation.
Addresses 351 Donner Laboratory, Lie Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA or: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; e-mail: [email protected]
Current Opinion in Cell Biology 1996, 6:369-373 0 Current Biology Ltd ISSN 0955-0674 Abbreviations chromatin assembly factor-l CAF-I histone acetyltransferase HAT nucleosome assembly protein-l NAP-I
Introduction The basic building block of the eukaryotic chromosome is the nucleosome. Nucleosomes consist of 146 bp of double-stranded DNA wrapped around two molecules each of histones HZA, H2B, H3 and H4 (see [1,2] for review). The majority of the genome is packaged into nucleosomes. Therefore, formation of new chromosomes during the S phase of the cell cycle requires existing nucleosomes to be transferred to the daughter strands and an equal quantity of new nucleosomes to be assembled. Transfer of existing nucleosomes to daughter DNA strands can be recapitulated in &-a, and can occur in the absence of factors other than DNA replication proteins (reviewed in [3,4-l). In other words, replication fork passage itself appears to induce transfer of existing nucleosomes to daughter DNA strands.
Chromatin assembly factor-l Numerous researchers over the years have fractionated cellular extracts in order to identify proteins that mediate the assembly of nucleosomes (see [3] for review). Most of the factors discovered assemble nucleosomes without dependence on DNA replication, and do not discriminate between newly synthesized histones and histones isolated from chromosomes. In contrast, chromatin assembly factor-I (CAF-I) is a protein complex that acts with both these specificities. CAF-I was first detected and purified from human cell nuclei during the study of in v&o SV40 DNA replication [ 11,121. Addition of CAF-I to SV40 replication reactions results in nucleosome assembly preferentially on replicating templates. Furthermore, CAF-I specifically uses histones present in human cell cytosolic extracts and will not deposit core histones purified from nuclear chromatin. This reaction occurs in two steps, as observed in r~,iero:CAF-I deposits newly synthesized histones H3 and H4 onto DNA during DNA replication, whereas H2A and H2B can bind subsequently to complete the nucleosome [13]. Thus, in vitro nucleosome assembly by CAF-I displays the specificities expected for a factor that operates primarily during S phase. The manner in which CAF-I distinguishes replicated DNA and newly synthesized histones, however, is not understood, and other proteins important for CAF-I action may remain to be discovered.
Mechanism This review focuses on the assembly of new nucleosomes. DNA and the core histones cannot self-assemble into nucleosomes under physiological conditions; hence de nwo nucleosome assembly requires assembly factors. In both yeast and mammalian cells, the majority of histone proteins are synthesized in a cell cycle dependent manner during S phase [5,6]. Histones are deposited onto the newly replicated DNA very soon after passage of the DNA replication fork [7,8]. A tetramer comprising two copies each of histones H3 and H4 is deposited onto DNA first;
of CAF-I action
Studies of CAF-I in the past year have involved the use of molecular, biochemical and cell biological techniques. The genes encoding the CAF-I ~150 and p60 subunits have been cloned and the subunits expressed as recombinant proteins [14**]. Recombinant ~150 and p60 function in etitro to assemble nucleosomes in a replication-preferential manner. Another study [lS**] showed that CAF-I ~150 and p60 subunits localize to punctate ‘replication foci’ as detected by bromodeoxyuridine labeling of nascent replicated DNA.
370
Nucleus and gene expression
As CAF-I is responsible for the deposition of histones H3 and H4 onto DNA [13], the involvement of CAF-I in nucleosome assembly during S phase predicted that CAF-I would be found in a complex with newly synthesized histones H3 and H4. This is indeed the case [14"°]. Antibodies specific for acetylated forms of histones H4 co-immunoprecipitate CAF-I subunits from cellular extracts. More importantly, anti-CAF-I antibodies co-immunoprecipitate the labeled, newly synthesized histones H3 and H4 from extracts of pulse-labeled cells. Biochemical activities similar to that of human CAF-I have been detected in Xenopus and Drosophila extracts [16,17"]. T h e s e activities function in a SV40 DNA replication reaction catalyzed by human proteins. Both recombinant human CAF-I and a partially purified Drosophila fraction were recently shown to assemble histones onto DNA that has undergone replication but is not being actively replicated [17"]. In this experiment, nucleosomes were assembled when CAF-I was added to reactions after DNA polymerases were inhibited by addition of aphidicolin. However, addition of aphidicolin before the replication reaction prevented later assembly by CAF-I. This suggests that an event required for CAF-I activity occurs durin& movement of the DNA replication fork. Furthermore, the ability of CAF-I to trigger nucleosome assembly after addition of aphidicolin decayed with time. A model is presented in Figure 1 in which CAF-I activity is triggered by labile structures left in the wake of replication forks. These could be composed of nucleic acid (e.g. single stranded DNA regions or R N A - D N A hybrids), proteins, or both. Another study of Drosophila CAF-I suggests that it can assemble nucleosomes in a pathway unlinked to D N A replication [18]. In this work, Drosophila embryo extracts were fractionated and assayed for activities that assemble nucleosomes onto topologically relaxed, non-replicating plasmids in the presence of core histones and Mg.ATP. Together, two chromatographic fractions caused rapid assembly of long arrays of regularly spaced nucleosomes, although some assembly was observed with either fraction alone. One fraction, termed dCAF4, is primarily composed of a 56kDa protein which binds to core histones. T h e other fraction, termed dCAF1, contains a replication-preferential assembly activity that functions in the human SV40 system. It is still, however, unclear if the proteins in the dCAF1 fraction responsible for the non-replication-linked assembly are the same as the factor that assembles nucleosomes during DNA replication. Complete purification and cloning of the relevant factors are required to address this issue. This approach should also determine if the dCAF1 factor is structurally, in addition to functionally, similar to its human counterpart. It will also be interesting to determine why the nonreplication-linked assembly reaction [18] is stimulated by ATP. Human CAF-I subunits do not contain nucleotide-
Figure 1
Leading
strand
andS"
Lagging
str
D
I
CAF-I
c
I,
CAF-I
c
© 1996 Current Opinion in Cell
Biology
Recognition of replicated DNA by CAF-I. The DNA synthesis proteins (black oval) at a DNA replication fork generate an uncharacterized species labeled '?' which is recognized by the complex of CAF-I and newly synthesized molecules of histones H3 and H4. The synthesis-related acetylation of H4 is indicated by 'Ac'. The '?' species is shown on both daughter strands because CAF-I assembles nucleosomes onto both daughter molecules. Nucleosomes existing prior to replication fork passage are omitted for clarity. The labile nature of '?' is indicated by the moving of '?' from the leading and lagging strands of the DNA; this was shown experimentally by the destruction of the ability of CAF-I to assemble nucleosomes onto replicated DNA after prolonged incubation of replication intermediates. See text and [17 °] for details.
binding consensus sequences [14"], nor do non-replicationlinked assembly factors such as nucleosome assembly protein-I, nucleoplasmin or N1/N2 [19-22]. In contrast, proteins involved in disruption or remodeling of existing nucleosomes, such as the SWI2 protein of yeast [23] or humans [24,25] and the related ISWI protein of Drosophila [26], do have such motifs, but these factors are not known to be involved in nucleosome assembly. Biochemistry HAT tricks
of histone
acetyltransferase:
A major limitation in the study of histone acetylation has been the lack of any completely purified acetyltransferase (or deacetylase) enzymes, genes encoding these enzymes, or cells lacking these functions. This situation has changed in the past year. Novel 'in-gel' assays have led to the identification of a chromatin-associated Tetrahymena acetyltransferase [27"]. Such methods should aid in the complete purification and cloning of acetyltransferases from various sources in the future. Another important advance has been the use of a chemical deblocking technique which allows direct sequence analysis of histone amino-terminal acetylation [28°]. This technique was used to show that newly synthesized histone H4 from both Drosophila and human cells is diacetylated on the
Nucleosome assembly: the CAF and the HAT Kaufman
same residues, LysS and LyslZ [29”]. This phenomenon is conserved in ciliates [29**,30].
biological
Working with Saccharomyces certwisiae, Sternglanz and coworkers [31”] cloned the first known gene encoding a histone acetyltransferase, termed HATl. In vitro, the Hatlp activity most strongly acetylates lysine 12 of histone H4, one of the residues identified as a site of acetylation in newly synthesized H4 [29”]. This finding has not yet, however, translated into understanding of the role of histone H4 acetylation in nucleosome assembly in vivo: deletion of the HATf gene does not cause any observed phenotype, except for a reduction of total histone acetyltransferase activity in extracts of the mutant strain. As this reduction is only 40%, a likely hypothesis is that there are multiple enzymes that can catalyze this reaction. The amino-terminal tails of other core histones undergo acetylation, so other HAT enzymes with different specificities must also exist.
Nucleosome assembly protein-l: biochemists caught NAPping? Study of nucleosome assembly has been beset by artefacts. For example, one early search for nucleosome assembly factors in Dt-osophda extracts led to the discovery that RNA can act in this role in vitro [32]. RNA, like other acidic polymers such as polyglutamic acid [33], is able to neutralize the strong positive charges of the histones and provide a way to achieve formation of nucleosomes under physiological conditions. Not surprisingly, proteins isolated as molecules that bind histones and assemble nucleosomes are generally highly acidic, or at least have acidic clusters of amino acids. One such factor that has gained recent attention is known as nucleosome assembly protein-I (NAP-I). NAP-I was first isolated as a protein that assembles nucleosomes onto a non-replicating DNA template [34]. Genes encoding NAP-I are evolutionarily conserved and have been cloned from humans [35], yeast [19,36*], and, more recently, from Xenopus [36*].
371
through M phase. These experiments are complicated by the fact that the multiple B-type cyclins have overlapping functions, but strains can be engineered that survive with the CLBZ gene as the gene encoding the only intact B-type cyclin responsible for M-phase progression. The authors clearly show that such Clb2-dependent strains are delayed in progression through M phase if the NAP2 gene is deleted [37*]. In other studies, immunolocalization experiments showed that yeast NAP-I is predominantly cytoplasmic (and unpublished data were cited claiming the same for vertebrate NAP-I [36’]; however, NAP-I was previously reported to be found instead in the nuclei of a transformed human cell line [38]). Yeast cells lacking NAP-I are resistant to cold temperatures and the microtubule-depolymerizing drug benomyl [37’], phenotypes usually associated with defects in microtubule dynamics. Together, these data at the least argue that part of the in viva function of NAP-I has nothing to do with nucleosome assembly. However, the data presented do not rule out the possibility that NAP-I could be a bifunctional protein, or that it never enters the yeast nucleus. Yeast cells lacking NAP-I are viable. However, no analysis of the cellular chromatin of these cells has been preseted to date. Unpublished data cited by Kellogg et al. [36’] suggest that, at least in Xenopus, there are multiple related NAPI-like genes, potentially making phenotypic interpretation in yeast difficult if functional redundancy exists in this organism. What does this example tell us about nucleosome assembly? As nucleosome assembly factors have been studied almost exclusively by biochemical techniques, it is important to proceed with caution until genetic data supporting the hypothesis that a particular protein acts in this process in vivo exist. Study of nucleosome assembly in genetically tractable organisms will therefore be essential in future years.
Conclusions What is the role of NAP-I in viva? The best evidence to date for the role of NAP-I comes from an unsuspected source and suggests that NAP-I is important for cell cycle progression rather than nucleosome assembly. Kellogg etal. [36*] and Kellogg and Murray [37*] isolated the NAP-I protein by affinity chromatography when extracts of S. cerevisiae or Xenopus were fractionated over columns of the yeast cyclin Clb2 protein (Clb2p) or the Xenopus cyclin B2 protein, respectively. Further experiments showed that these physical interactions are unlikely to be in vitro artefacts. First, the cyclin B-NAP-I interaction is specific and occurs both in vitro and in cellular extracts; that is, NAP-I co-immunoprecipitates with yeast Clb2p, but not with another B-type cyclin, Clb3p [36*]. Second, and more importantly, genetic evidence shows that NAP-I is required for Clb2p to exert its full range of effects on the budding yeast cell cycle 137.1. Clb2p (together with other B-type cyclins) plays a major role in progression
The past year has seen the development of important reagents for the study of chromatin assembly, but the major questions remain. For example, which proteins are required for the assembly of new nucleosomes in &JO? Although biochemical data suggest an important role for CAF-I, they do not formally prove its requirement in vivo. Also, CAF-I specifically uses modified histones that are present in minute amounts in cellular extracts. This has made it difficult to establish an assay system for CAF-I that uses defined components. Molecular cloning and overexpression of the human CAF-I subunits will, however, facilitate detailed mechanistic study in the future. The role of the acetylation of histone H4 (and H3 in some species) that occurs on newly synthesized molecules is unclear. Does this post-translational modification serve as a tag for trafficking histones from the cytoplasm to the
372
Nucleus
and gene expression
nucleus or for subsequent localization to replication foci, or for another, as yet unperceived, function? A combination of biochemical, genetic and cell biological techniques will be required to answer these questions.
Note added in proof The in-gel assay described in [27*] has now resulted in the cloning of the first gene encoding a nuclear, chromatin-bound histone acetyltransferase [39]. This gene encodes a protein which is homologous to the yeast protein encoded by GCN5 that is required for the full function of other transcriptional activator proteins. The authors of [39] show that a recombinant GCNS protein possesses HAT activity. This work establishes the first direct link between histone acetylation and gene activation.
Acknowledgements I thank Kathleen Collins, Rohinton Kamakaka and Aiain Verrcauit for comments on the manuscript and Bruce Stillman for advice and support during my postdoctoral studies. I have been supported as a Smith Kline Beecham Fellow of the Life Sciences Research Foundation and by grant CA13106 from the National Cancer Institute.
This paper describes the molecular cloning of the genes encoding the ~150 and p60 subunits of human CAF-I. ~150 contains both a large cluster highly enriched in positively and negatively charged residues, and smaller acidic patches. p80 is a member of the WD-repeat family of proteins. Both subunits are required for replication-preferential chromatin assembly activity during SV40 DNA reolication reactions in vitro. lmmunoorecioitation exoeriments using pulse-ladeled cellular extracts show that neily syhthesized &lecules of histones H3 and H4 are associated with the CAF subunits in human cell extracts. Also, addition of recombinant ~150 alone, but not p80 alone, to human cytosolic extracts allows for subsequent immunoprecipitation of the newly synthesized histones from the extract by using anti-CAF antibodies, suggesting that it is pi 50 that is responsible for interacting with the histones. The reagents generated in this work, together with new tools for the study of histone acetylation obtained from other laboratories [27*,28*,31 **I, will be important for eventually understanding how CAF-I specifically selects newly synthesized histones for deposition onto newly replicated DNA. 15. ..
In this work, the author used monocional antibodies raised against the ~150 and p80 subunits of human CAF-I ([40] describes how these antibodies were raised) for immunolocalization of these subunits. Human HeLa cell nuclei were incubated with bromodeoxyuridine to mark sites of DNA replication prior to fixation. The CAF subunits colocalized with small, punctate ‘replication foci’, that is, sites of incorporation of deoxynucleotides in S-phase nuclei. The single-strand-binding protein Replication Protein A (RPA) also localized at these sites, and also to other sites; the latter localization may be due to the role of RPA in processes other than DNA replication, such as DNA repair and recombination. The data support the contention that CAF-I acts during DNA replication to assemble nucleosomes. 18.
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Brownell JE, Allis CD: An activity gel assay detects a single, catslytlcelly active histone acetyltransferase subunit in Tetrahymene macronuclel. froc Nat/ Acad Sci USA 1995, 92:6364-6368. This paper describes an important technical innovation that will aid in the detection and purification of acetyitransferase enzymes In thii ‘in-gel’ approach, core histone proteins are polymerized directly into SDS-polyacrylamide gels. After electrophoresis and denaturationlrenaturation steps, the gels are soaked in radioactive acetyl coenzyme A. The renatured acetyltransferase enzyme then transfers label to the histones. Autoradiography detects the position of the enzyme in the gel. The authors employ this technique to follow significant purification of a histone acetyltransferase from ktrahymena chromatin. Similar application should allow analysis of acetyltransferases from other sources, provided that the enzymes are able to withstand the denaturing conditions encountered during electrophoresis and other treatments.
27. .
Sobel RE, Cook RG, Allis CD: Non-random acetytatlon of hlstone H4 by a cytopiasmlc histone acetyltransferase as determined by novel methodology. J Biol Chem 1994, 289:18576-l 8582. Previous attempts to determine the nature of post-translational modifications near the amino termini of histone proteins by Edman degradation sequence analysis were prevented in many cases by chemical blockage of the aminoterminal a-amino group. To circumvent this, the authors exploit a chemical deblocking technique that allows subsequent protein sequencing. This deblocking technique is used to show that a cytosolic fraction from Drosophila embryos that contains a ‘type B’ (non-chromatin bound) histone acetyltransferase specifically modifies lysine 12 of Drosophila histone H4, in addition to the analogous lysine 11 of Tetrahymena histone H4. Sobel RE, Cook RG, Perry CA, Annunziato AT, Allis CD: Conservation of deposition-related acetylatlon sites In newly synthesized histones H3 and H4. Proc Nat/ Acad Sci USA 1995,82:1237-l 241. Using the technique to deblock the amino termini of histone proteins described in [28’], the authors demonstrate that newly synthesized histone H4 is diacetylated on lysines 5 and 12 in human HeLa cells and in Drosophila cells. Together with previous data showing that the analogous lysines 4 and 11 are modified in Tetrahymena [28’,301, a remarkable evolutionary conservation is demonstrated. However, this paper [29’*] also shows that the same is not true for newly synthesized histone H3. Different lysines in the amino-terminal region of histone H3 are acetylated in ktrahymena and Drosophila, and no acetylation was detected in human H3. 30.
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Kleff S, Andruis ED, Anderson CW, Sternglanz R: identification of a gene encoding a yeast histone H4 acetyltransferase. J Biol Chem 1995, 270:24674-24677. This paper describes the first isolation of a gene encoding a histone acetyltransferase that modifies the e-amino groups of lysines (rather than the a-amino group on the amino terminus of the polypeptide chain). This S. cerevisiae gene, known as HATl, was cloned using a combined genetic/biochemical approach. Chromatographic fractions of yeast cell extracts from individual highly mutagenized strains were assayed for the ability to transfer labeled acetyl groups to a peptide containing amino-terminal residues of histone H4. A strain was found that demonstrated a 40% decrease in label incorporation in this assay; multiple backcrossing of the strain to wild-type cells showed that the defect was caused by a mutation at a single locus which was then genetically mapped. The HAT1 gene was then identified from genomic DNA fragments of that locus on the basis of its ability
the CAF and the HAT Kaufman
373
to restore acetyltransferase activity to the mutant cells. Protein sequencing of peptides acetylated in vitro by Hat1 p shows that the enzyme preferentially acetylates lysine 12 of histone H4, one of the residues known to be acetylated on newly synthesized histone molecules (see [29*1). However, this paper raises more questions than it answers, because neither the original mutation nor complete disruption of HAT7 causes any known phenotype other than the biochemical defect. Because hat7 mutant cells still have 6096 of the wild-type level of histone H4 acetyltransferase activity, it is likely that there are multiple redundant pathways involved in histone acetylation. 32.
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28. .
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Kellogg DR, Kikuchi A, Fujii-Nakata T, Turck CW, Murray AW: Members of the NAP/SET family of proteins interact specifically with B-type cyclins. J Cell Biol 1995, 130:661-673. This paper, together with 137.1, serves as an intriguing example of how genetic and cell biological analyses need to be used in the study of chromatin assembly. In a search for proteins that interact with the B-type cyclins that are important for progression through the M phase of the cell cycle, the authors isolated a previously described protein called NAP-I (see (19,341) from extracts of both yeast and Xenopus. The authors provide biochemical, genetic and cell biological data to show that yeast NAP-l is required for the cyclin Clb2 to perform its full range of functions in coordinating the cell cycle. One unanswered question, therefore, is whether NAP-I has any role in chromatin assembly in viva. Alternatively, its activity as a nucleosomeassembly protein could be a biochemical artefact owing to its ability to serve as an acidic polymer like RNA [32] or polyglutamic acid [33]. As genetic data suggest that the NAP7 gene is required for microtubule dynamics, and biochemical data show that NAP-I binds both to core histones 1411 and to cyclin B, another possibility is that the NAP-I family of proteins is involved in coordinating interactions between chromosomes, microtubules, and cyclin B during mitosis. 36. .
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31. ..
Nucleosome assembly: the CAF and the HAT Paul D Kaufman Recent data argue strongly that a protein complex termed chromatin assembly factor-l (CAF-I) plays a major role in de nova nucleosome assembly during DNA replication. Human CAF-I deposits newly synthesized, acetylated histones onto replicated DNA in vitro and localizes to sites of DNA replication in S-phase cells. Specific lysines of the histones used for nucleosome assembly are acetylated; in the past year the first gene encoding a histone acetyltransferase was cloned. However, mechanistic links between histone acetylation and nucleosome assembly have not been established in vivo or in vitro.
two H2A-H2B heterodimers complete the nucleosome.
are added
subsequently
to
E-amino groups of lysines near the amino-terminal regions of histones undergo reversible acetylation (reviewed in [9,10]). This modification can occur during two distinct biological processes. First, histones already incorporated into nuclear chromatin can become acetylated. Second, a different pattern of acetylation is observed on histones that are newly synthesized in the cytoplasm. This review, which concerns assembly of newly synthesized histones into nucleosomes, will focus on the latter type of acetylation.
Addresses 351 Donner Laboratory, Lie Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA or: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; e-mail: [email protected]
Current Opinion in Cell Biology 1996, 6:369-373 0 Current Biology Ltd ISSN 0955-0674 Abbreviations chromatin assembly factor-l CAF-I histone acetyltransferase HAT nucleosome assembly protein-l NAP-I
Introduction The basic building block of the eukaryotic chromosome is the nucleosome. Nucleosomes consist of 146 bp of double-stranded DNA wrapped around two molecules each of histones HZA, H2B, H3 and H4 (see [1,2] for review). The majority of the genome is packaged into nucleosomes. Therefore, formation of new chromosomes during the S phase of the cell cycle requires existing nucleosomes to be transferred to the daughter strands and an equal quantity of new nucleosomes to be assembled. Transfer of existing nucleosomes to daughter DNA strands can be recapitulated in &-a, and can occur in the absence of factors other than DNA replication proteins (reviewed in [3,4-l). In other words, replication fork passage itself appears to induce transfer of existing nucleosomes to daughter DNA strands.
Chromatin assembly factor-l Numerous researchers over the years have fractionated cellular extracts in order to identify proteins that mediate the assembly of nucleosomes (see [3] for review). Most of the factors discovered assemble nucleosomes without dependence on DNA replication, and do not discriminate between newly synthesized histones and histones isolated from chromosomes. In contrast, chromatin assembly factor-I (CAF-I) is a protein complex that acts with both these specificities. CAF-I was first detected and purified from human cell nuclei during the study of in v&o SV40 DNA replication [ 11,121. Addition of CAF-I to SV40 replication reactions results in nucleosome assembly preferentially on replicating templates. Furthermore, CAF-I specifically uses histones present in human cell cytosolic extracts and will not deposit core histones purified from nuclear chromatin. This reaction occurs in two steps, as observed in r~,iero:CAF-I deposits newly synthesized histones H3 and H4 onto DNA during DNA replication, whereas H2A and H2B can bind subsequently to complete the nucleosome [13]. Thus, in vitro nucleosome assembly by CAF-I displays the specificities expected for a factor that operates primarily during S phase. The manner in which CAF-I distinguishes replicated DNA and newly synthesized histones, however, is not understood, and other proteins important for CAF-I action may remain to be discovered.
Mechanism This review focuses on the assembly of new nucleosomes. DNA and the core histones cannot self-assemble into nucleosomes under physiological conditions; hence de nwo nucleosome assembly requires assembly factors. In both yeast and mammalian cells, the majority of histone proteins are synthesized in a cell cycle dependent manner during S phase [5,6]. Histones are deposited onto the newly replicated DNA very soon after passage of the DNA replication fork [7,8]. A tetramer comprising two copies each of histones H3 and H4 is deposited onto DNA first;
of CAF-I action
Studies of CAF-I in the past year have involved the use of molecular, biochemical and cell biological techniques. The genes encoding the CAF-I ~150 and p60 subunits have been cloned and the subunits expressed as recombinant proteins [14**]. Recombinant ~150 and p60 function in etitro to assemble nucleosomes in a replication-preferential manner. Another study [lS**] showed that CAF-I ~150 and p60 subunits localize to punctate ‘replication foci’ as detected by bromodeoxyuridine labeling of nascent replicated DNA.
370
Nucleus and gene expression
As CAF-I is responsible for the deposition of histones H3 and H4 onto DNA [13], the involvement of CAF-I in nucleosome assembly during S phase predicted that CAF-I would be found in a complex with newly synthesized histones H3 and H4. This is indeed the case [14"°]. Antibodies specific for acetylated forms of histones H4 co-immunoprecipitate CAF-I subunits from cellular extracts. More importantly, anti-CAF-I antibodies co-immunoprecipitate the labeled, newly synthesized histones H3 and H4 from extracts of pulse-labeled cells. Biochemical activities similar to that of human CAF-I have been detected in Xenopus and Drosophila extracts [16,17"]. T h e s e activities function in a SV40 DNA replication reaction catalyzed by human proteins. Both recombinant human CAF-I and a partially purified Drosophila fraction were recently shown to assemble histones onto DNA that has undergone replication but is not being actively replicated [17"]. In this experiment, nucleosomes were assembled when CAF-I was added to reactions after DNA polymerases were inhibited by addition of aphidicolin. However, addition of aphidicolin before the replication reaction prevented later assembly by CAF-I. This suggests that an event required for CAF-I activity occurs durin& movement of the DNA replication fork. Furthermore, the ability of CAF-I to trigger nucleosome assembly after addition of aphidicolin decayed with time. A model is presented in Figure 1 in which CAF-I activity is triggered by labile structures left in the wake of replication forks. These could be composed of nucleic acid (e.g. single stranded DNA regions or R N A - D N A hybrids), proteins, or both. Another study of Drosophila CAF-I suggests that it can assemble nucleosomes in a pathway unlinked to D N A replication [18]. In this work, Drosophila embryo extracts were fractionated and assayed for activities that assemble nucleosomes onto topologically relaxed, non-replicating plasmids in the presence of core histones and Mg.ATP. Together, two chromatographic fractions caused rapid assembly of long arrays of regularly spaced nucleosomes, although some assembly was observed with either fraction alone. One fraction, termed dCAF4, is primarily composed of a 56kDa protein which binds to core histones. T h e other fraction, termed dCAF1, contains a replication-preferential assembly activity that functions in the human SV40 system. It is still, however, unclear if the proteins in the dCAF1 fraction responsible for the non-replication-linked assembly are the same as the factor that assembles nucleosomes during DNA replication. Complete purification and cloning of the relevant factors are required to address this issue. This approach should also determine if the dCAF1 factor is structurally, in addition to functionally, similar to its human counterpart. It will also be interesting to determine why the nonreplication-linked assembly reaction [18] is stimulated by ATP. Human CAF-I subunits do not contain nucleotide-
Figure 1
Leading
strand
andS"
Lagging
str
D
I
CAF-I
c
I,
CAF-I
c
© 1996 Current Opinion in Cell
Biology
Recognition of replicated DNA by CAF-I. The DNA synthesis proteins (black oval) at a DNA replication fork generate an uncharacterized species labeled '?' which is recognized by the complex of CAF-I and newly synthesized molecules of histones H3 and H4. The synthesis-related acetylation of H4 is indicated by 'Ac'. The '?' species is shown on both daughter strands because CAF-I assembles nucleosomes onto both daughter molecules. Nucleosomes existing prior to replication fork passage are omitted for clarity. The labile nature of '?' is indicated by the moving of '?' from the leading and lagging strands of the DNA; this was shown experimentally by the destruction of the ability of CAF-I to assemble nucleosomes onto replicated DNA after prolonged incubation of replication intermediates. See text and [17 °] for details.
binding consensus sequences [14"], nor do non-replicationlinked assembly factors such as nucleosome assembly protein-I, nucleoplasmin or N1/N2 [19-22]. In contrast, proteins involved in disruption or remodeling of existing nucleosomes, such as the SWI2 protein of yeast [23] or humans [24,25] and the related ISWI protein of Drosophila [26], do have such motifs, but these factors are not known to be involved in nucleosome assembly. Biochemistry HAT tricks
of histone
acetyltransferase:
A major limitation in the study of histone acetylation has been the lack of any completely purified acetyltransferase (or deacetylase) enzymes, genes encoding these enzymes, or cells lacking these functions. This situation has changed in the past year. Novel 'in-gel' assays have led to the identification of a chromatin-associated Tetrahymena acetyltransferase [27"]. Such methods should aid in the complete purification and cloning of acetyltransferases from various sources in the future. Another important advance has been the use of a chemical deblocking technique which allows direct sequence analysis of histone amino-terminal acetylation [28°]. This technique was used to show that newly synthesized histone H4 from both Drosophila and human cells is diacetylated on the
Nucleosome assembly: the CAF and the HAT Kaufman
same residues, LysS and LyslZ [29”]. This phenomenon is conserved in ciliates [29**,30].
biological
Working with Saccharomyces certwisiae, Sternglanz and coworkers [31”] cloned the first known gene encoding a histone acetyltransferase, termed HATl. In vitro, the Hatlp activity most strongly acetylates lysine 12 of histone H4, one of the residues identified as a site of acetylation in newly synthesized H4 [29”]. This finding has not yet, however, translated into understanding of the role of histone H4 acetylation in nucleosome assembly in vivo: deletion of the HATf gene does not cause any observed phenotype, except for a reduction of total histone acetyltransferase activity in extracts of the mutant strain. As this reduction is only 40%, a likely hypothesis is that there are multiple enzymes that can catalyze this reaction. The amino-terminal tails of other core histones undergo acetylation, so other HAT enzymes with different specificities must also exist.
Nucleosome assembly protein-l: biochemists caught NAPping? Study of nucleosome assembly has been beset by artefacts. For example, one early search for nucleosome assembly factors in Dt-osophda extracts led to the discovery that RNA can act in this role in vitro [32]. RNA, like other acidic polymers such as polyglutamic acid [33], is able to neutralize the strong positive charges of the histones and provide a way to achieve formation of nucleosomes under physiological conditions. Not surprisingly, proteins isolated as molecules that bind histones and assemble nucleosomes are generally highly acidic, or at least have acidic clusters of amino acids. One such factor that has gained recent attention is known as nucleosome assembly protein-I (NAP-I). NAP-I was first isolated as a protein that assembles nucleosomes onto a non-replicating DNA template [34]. Genes encoding NAP-I are evolutionarily conserved and have been cloned from humans [35], yeast [19,36*], and, more recently, from Xenopus [36*].
371
through M phase. These experiments are complicated by the fact that the multiple B-type cyclins have overlapping functions, but strains can be engineered that survive with the CLBZ gene as the gene encoding the only intact B-type cyclin responsible for M-phase progression. The authors clearly show that such Clb2-dependent strains are delayed in progression through M phase if the NAP2 gene is deleted [37*]. In other studies, immunolocalization experiments showed that yeast NAP-I is predominantly cytoplasmic (and unpublished data were cited claiming the same for vertebrate NAP-I [36’]; however, NAP-I was previously reported to be found instead in the nuclei of a transformed human cell line [38]). Yeast cells lacking NAP-I are resistant to cold temperatures and the microtubule-depolymerizing drug benomyl [37’], phenotypes usually associated with defects in microtubule dynamics. Together, these data at the least argue that part of the in viva function of NAP-I has nothing to do with nucleosome assembly. However, the data presented do not rule out the possibility that NAP-I could be a bifunctional protein, or that it never enters the yeast nucleus. Yeast cells lacking NAP-I are viable. However, no analysis of the cellular chromatin of these cells has been preseted to date. Unpublished data cited by Kellogg et al. [36’] suggest that, at least in Xenopus, there are multiple related NAPI-like genes, potentially making phenotypic interpretation in yeast difficult if functional redundancy exists in this organism. What does this example tell us about nucleosome assembly? As nucleosome assembly factors have been studied almost exclusively by biochemical techniques, it is important to proceed with caution until genetic data supporting the hypothesis that a particular protein acts in this process in vivo exist. Study of nucleosome assembly in genetically tractable organisms will therefore be essential in future years.
Conclusions What is the role of NAP-I in viva? The best evidence to date for the role of NAP-I comes from an unsuspected source and suggests that NAP-I is important for cell cycle progression rather than nucleosome assembly. Kellogg etal. [36*] and Kellogg and Murray [37*] isolated the NAP-I protein by affinity chromatography when extracts of S. cerevisiae or Xenopus were fractionated over columns of the yeast cyclin Clb2 protein (Clb2p) or the Xenopus cyclin B2 protein, respectively. Further experiments showed that these physical interactions are unlikely to be in vitro artefacts. First, the cyclin B-NAP-I interaction is specific and occurs both in vitro and in cellular extracts; that is, NAP-I co-immunoprecipitates with yeast Clb2p, but not with another B-type cyclin, Clb3p [36*]. Second, and more importantly, genetic evidence shows that NAP-I is required for Clb2p to exert its full range of effects on the budding yeast cell cycle 137.1. Clb2p (together with other B-type cyclins) plays a major role in progression
The past year has seen the development of important reagents for the study of chromatin assembly, but the major questions remain. For example, which proteins are required for the assembly of new nucleosomes in &JO? Although biochemical data suggest an important role for CAF-I, they do not formally prove its requirement in vivo. Also, CAF-I specifically uses modified histones that are present in minute amounts in cellular extracts. This has made it difficult to establish an assay system for CAF-I that uses defined components. Molecular cloning and overexpression of the human CAF-I subunits will, however, facilitate detailed mechanistic study in the future. The role of the acetylation of histone H4 (and H3 in some species) that occurs on newly synthesized molecules is unclear. Does this post-translational modification serve as a tag for trafficking histones from the cytoplasm to the
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Nucleus
and gene expression
nucleus or for subsequent localization to replication foci, or for another, as yet unperceived, function? A combination of biochemical, genetic and cell biological techniques will be required to answer these questions.
Note added in proof The in-gel assay described in [27*] has now resulted in the cloning of the first gene encoding a nuclear, chromatin-bound histone acetyltransferase [39]. This gene encodes a protein which is homologous to the yeast protein encoded by GCN5 that is required for the full function of other transcriptional activator proteins. The authors of [39] show that a recombinant GCNS protein possesses HAT activity. This work establishes the first direct link between histone acetylation and gene activation.
Acknowledgements I thank Kathleen Collins, Rohinton Kamakaka and Aiain Verrcauit for comments on the manuscript and Bruce Stillman for advice and support during my postdoctoral studies. I have been supported as a Smith Kline Beecham Fellow of the Life Sciences Research Foundation and by grant CA13106 from the National Cancer Institute.
This paper describes the molecular cloning of the genes encoding the ~150 and p60 subunits of human CAF-I. ~150 contains both a large cluster highly enriched in positively and negatively charged residues, and smaller acidic patches. p80 is a member of the WD-repeat family of proteins. Both subunits are required for replication-preferential chromatin assembly activity during SV40 DNA reolication reactions in vitro. lmmunoorecioitation exoeriments using pulse-ladeled cellular extracts show that neily syhthesized &lecules of histones H3 and H4 are associated with the CAF subunits in human cell extracts. Also, addition of recombinant ~150 alone, but not p80 alone, to human cytosolic extracts allows for subsequent immunoprecipitation of the newly synthesized histones from the extract by using anti-CAF antibodies, suggesting that it is pi 50 that is responsible for interacting with the histones. The reagents generated in this work, together with new tools for the study of histone acetylation obtained from other laboratories [27*,28*,31 **I, will be important for eventually understanding how CAF-I specifically selects newly synthesized histones for deposition onto newly replicated DNA. 15. ..
In this work, the author used monocional antibodies raised against the ~150 and p80 subunits of human CAF-I ([40] describes how these antibodies were raised) for immunolocalization of these subunits. Human HeLa cell nuclei were incubated with bromodeoxyuridine to mark sites of DNA replication prior to fixation. The CAF subunits colocalized with small, punctate ‘replication foci’, that is, sites of incorporation of deoxynucleotides in S-phase nuclei. The single-strand-binding protein Replication Protein A (RPA) also localized at these sites, and also to other sites; the latter localization may be due to the role of RPA in processes other than DNA replication, such as DNA repair and recombination. The data support the contention that CAF-I acts during DNA replication to assemble nucleosomes. 18.
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to restore acetyltransferase activity to the mutant cells. Protein sequencing of peptides acetylated in vitro by Hat1 p shows that the enzyme preferentially acetylates lysine 12 of histone H4, one of the residues known to be acetylated on newly synthesized histone molecules (see [29*1). However, this paper raises more questions than it answers, because neither the original mutation nor complete disruption of HAT7 causes any known phenotype other than the biochemical defect. Because hat7 mutant cells still have 6096 of the wild-type level of histone H4 acetyltransferase activity, it is likely that there are multiple redundant pathways involved in histone acetylation. 32.
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