Chromatin assembly factor 1 subunit


Biology of the CAF-I complex

Cac1p is a subunit of yeast chromatin assembly factor I (yCAF-I) that is thought to assemble nucleosomes containing diacetylated histones onto newly replicated DNA. Although cac1 delta cells can establish and maintain transcriptional repression at telomeres, they display a reduced heritability of the repressed state. Single-cell analysis reveals that individual cac1 delta cells switch from transcriptionally 'off' to transcriptionally 'on' more often per cell cycle than wild-type cells. In addition, cac1 delta cells are defective for transcriptional silencing near internal tracts of C(1-3)A sequence, but they show no defect in silencing at the silent mating type loci when analyzed by a reverse transcription-PCR assay. Despite the loss of transcriptional silencing at telomeres and internal C(1-3)A tracts, subtelomeric DNA is organized into nucleosomes that have all of the features characteristic of silent chromatin, such as hypoacetylation of histone H4 and protection from methylation by the E. coli dam methylase. Thus, these features of silent chromatin are not sufficient for stable maintenance of a silent chromatin state. It is proposed that the inheritance of the transcriptionally repressed state requires the specific pattern of histone acetylation conferred by yCAF-I-mediated nucleosome assembly (Monson, 1997).

A cell free system that supports replication-dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF-I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF-I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4-replicated DNA complex is a stable intermediate that exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF-I in at least the first step of the reaction suggests a level of cellular control for this fundamental process (Smith, 1991).

Circular single-stranded phage M13 DNA is used as a template for complementary strand synthesis in cytosolic extracts from proliferating HeLa cells. DNA synthesis is initiated by one priming event (or at a maximum, two), and typically leads to covalently closed double-stranded reaction products. When carried out in the presence of the nuclear chromatin assembly factor CAF-1, complementary strand synthesis is accompanied by nucleosome assembly. This novel system is very useful for the study of basic biochemical aspects concerning the assembly of nucleosomes. The activity of CAF-1 completely depends on complementary strand synthesis and acts stoichiometrically to promote the assembly of nucleosomes in a noncooperative manner. Apparently, CAF-1 activity is coupled to DNA synthesis via a structural feature of replicating DNA, most likely its partial single strandedness (Krude, 1993).

During the organization and acetylation of nascent histones prior to their stable incorporation into chromatin two somatic non-nucleosomal histone complexes are detected: one containing nascent H3 and H4, and a second containing H2A (and probably H2B) in association with the nonhistone protein NAP-1. The H3/H4 complex has a sedimentation coefficient of 5-6S, consistent with the presence of one or more escort proteins. H4 in the cytosolic H3/H4 complex is diacetylated, fully in accord with the acetylation state of newly synthesized H4 in chromatin. The diacetylation of nascent human H4 is therefore completed prior to nucleosome assembly. As part of the study of the nascent H3/H4 complex, the cytoplasmic histone acetyltransferase most likely responsible for acetylating newly synthesized H4 was also investigated. HeLa histone acetyltransferase B (HAT B) acetylates H4 but not H3 in vitro, and maximally diacetylates H4 even in the presence of sodium butyrate. Human HAT B acetylates H4 exclusively on the lysine residues at positions 5 and 12, in complete agreement with the highly conserved acetylation pattern of nascent nucleosomal H4, and has a native molecular weight of approximately 100 kDa. Based on these findings a model has been presented for the involvement of histone acetylation and NAP-1 in H2A/H2B deposition and exchange, during nucleosome assembly and chromatin remodeling in vivo (Chang, 1997).

In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage. A protein complex from human cells, termed chromatin assembly factor-I (CAF-I) assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro. Amino acid sequence data from purified yeast (Saccharomyces cerevisiae) CAF-I led to identification of the genes encoding each subunit in the yeast genome data base. The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively. The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and has been identified as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms. Thus, CAF-I has been conserved functionally and structurally from yeast to human cells. Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability. However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays. This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair. Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus. Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo (Kaufman, 1997).

One of the best studied examples of chromatin based gene silencing occurs at the HM loci in the budding yeast S cerevisiae. CAC1/RLF2 encodes the largest subunit of chromatin assembly factor I (CAF-I), a complex that assembles newly synthesized histones onto recently replicated DNA in vitro. Chromatin assembly factor I (CAF-I) preferentially assembles histones H3 and H4 with the acetylation pattern of newly synthesized cytoplasmic histones. Mating type genes expressed from the MAT locus determine the yeast mating type in haploid cells. Wild-type strains have functional but transcriptionally repressed mating information at the two HM loci, HML and HMR. Haploid cells normally respond to the mating pheromone of the opposite mating type by arresting in late G1 and forming mating projections (shmoos). The Sir complex of proteins comprises components of yeast heterochromatin that are involved in transcriptional silencing, and act by physical interaction with Histone H3 and H4. CAC1, encoding the largest subunit of CAF-I, is identical to RLF2, a gene that was identified in a screen for mutants defective in telomere-related functions. In vivo, cac1/rlf2 mutants are defective in telomeric silencing and they mislocalize Rap1p, a telomere-binding protein. In cells lacking CAF-I, the silent mating loci are partially derepressed. MATa cac1 cells exhibit an unusual response to alpha-factor: they arrest and initially form shmoos, but are unable to sustain the arrest state, giving rise to clusters of shmooing cells. cac1 MATa HMLa HMRa strains do not form these shmoo clusters, indicating that derepression of HMLalpha causes the shmoo cluster phenotype in cac1 cells. When SIR3 is reintroduced into sir1 sir3 cells, HML remains derepressed, indicating that SIR1 is required for the re-establishment of silencing at HML. In contrast, when SIR3 is reintroduced into cac1 sir3 cells, silencing is restored to HML, indicating that CAF-I is not required for the re-establishment of silencing. Loss of the other CAF-I subunits (Cac2p and Cac3p/Msi1p) also results in the shmoo cluster phenotype, implying that loss of CAF-I activity gives rise to this unstable repression of HMLalpha. Strains carrying certain mutations in the amino terminus of histone H4 and strains with limiting amounts of Sir2p or Sir3p also form shmoo clusters, implying that the shmoo cluster phenotype is indicative of defects in maintenance of the structural integrity of silent chromatin. MATa cac sir1 double mutants have a synergistic mating defect, suggesting that the two silencing mechanisms, establishment and maintenance, function cooperatively (Enomoto, 1998).

Chromatin assembly factor I (CAF-I) from human cell nuclei is a three-subunit protein complex that assembles histone octamers onto replicating DNA in a cell-free system. Sequences of cDNAs encoding the two largest CAF-I subunits reveal that the p150 protein contains large clusters of charged residues, whereas p60 contains WD repeats. p150 and p60 directly interact and are both required for DNA replication-dependent assembly of nucleosomes. Deletion of the p60-binding domain from the p150 protein prevents chromatin assembly. p150 and p60 form complexes with newly synthesized histones H3 and acetylated H4 in human cell extracts, suggesting that such complexes are intermediates between histone synthesis and assembly onto replicating DNA (Kaufman, 1995).

Conserved patterns of histone acetylation by histone acetyltransferase

During periods of active DNA replication and chromatin assembly, newly synthesized histone H4 is deposited in a diacetylated form. In Tetrahymena, a specific pair of residues, lysines 4 and 11, has been shown to undergo this modification in vivo. Presumably this reaction is catalyzed, at least in part, by histone acetyltransferases (HAT) of the B type, cytoplasmic enzymes displaying strong preference for free, non-chromatin-bound H4. To investigate which lysines are preferred acetylation sites in H4 in other organisms, a cytoplasmic HAT B activity was prepared from Drosophila embryos and used to acetylate H4 from several species. When either H4 or synthetic NH2-terminal peptides from Tetrahymena were used as unblocked substrates, direct microsequence analyses shows that [3H]acetate is preferentially incorporated at lysine 11 with little, if any, incorporation at other conserved, acetylatable lysines. Drosophila H4 was chemically deblocked following its acetylation in vitro using conditions that do not deacetylate internal lysines. Direct sequence analysis verifies the correct NH2-terminal sequence of Drosophila H4 and demonstrates that [3H]acetate incorporation occurs preferentially on lysine 12, the residue analogous to lysine 11 in Tetrahymena. These data show remarkable preference for lysine 11/12 by the Drosophila HAT B activity in vitro and provide support for the assertion that this activity functions to acetylate new H4, at least in part, for deposition and chromatin assembly in vivo. Since most H4s, like Drosophila, are blocked at their amino termini by an acetylthreonine or acetylserine, these results demonstrate that this deblocking and microsequencing strategy can be used to study acetylation site utilization in H4 and presumably other core histones that are NH2 terminally blocked with these residues (Sobel, 1994).

Newly synthesized histone H4 is deposited in a diacetylated isoform in a wide variety of organisms. In Tetrahymena a specific pair of residues, lysines 4 and 11, have been shown to undergo this modification in vivo. The analogous residues, lysines 5 and 12, are acetylated in Drosophila and HeLa H4. These data strongly suggest that deposition-related acetylation sites in H4 have been highly, perhaps absolutely, conserved. In Tetrahymena and Drosophila newly synthesized histone H3 is also deposited in several modified forms. Using pulse-labeled H3 it has been determined that, like H4, a specific but distinct subset of lysines is acetylated in these organisms. In Tetrahymena, lysines 9 and 14 are highly preferred sites of acetylation in new H3; in Drosophila, lysines 14 and 23 are strongly preferred. No evidence has been obtained for acetylation of newly synthesized H3 in HeLa cells. Thus, although the pattern and sites of deposition-related acetylation appear to be highly conserved in H4, the same does not appear to be the case for histone H3 (Sobel, 1995).

CAF-1, PCNA and gene silencing

Chromatin assembly factor 1 (CAF-1) is required for inheritance of epigenetically determined chromosomal states in vivo and promotes assembly of chromatin during DNA replication in vitro. After DNA replication, the replicated (but not unreplicated) DNA is also competent for CAF-1-dependent chromatin assembly. The proliferating cell nuclear antigen (PCNA), a DNA polymerase clamp, is a component of the replication-dependent marking of DNA for chromatin assembly. The clamp loader, replication factor C (RFC), can reverse this mark by unloading PCNA from the replicated DNA. PCNA binds directly to p150, the largest subunit of CAF-1, and the two proteins colocalize at sites of DNA replication in cells. It is suggested that PCNA and CAF-1 connect DNA replication to chromatin assembly and the inheritance of epigenetic chromosome states (Shibahara, 1999).

CAF-1 assembles nucleosomes during DNA replication in vitro on both the leading and lagging strands behind a replication fork. Nucleosome assembly is an ordered process, with histones H3 and H4 first loaded onto the DNA during replication in a CAF-1-dependent manner, and soon thereafter, histones H2A and H2B are added to form a mature nucleosome. This is most likely how the bulk of de novo nucleosome assembly occurs during S phase. In the current paper, however, it has been demonstrated that nucleosome assembly can be temporally dissociated from passage of the DNA replication fork. Addition of CAF-1 after completion of DNA replication allows nucleosome assembly on the replicated DNA, but not on the unreplicated DNA that is present in the same reaction. Thus, the replicated DNA is marked or imprinted for subsequent CAF-1-dependent processes. It has been suggested that a component of the DNA replication fork mediates CAF-1 function, and it now seems likely that PCNA is that factor (Shibahara, 1999 and references).

The PCNA clamp is involved in the synthesis of both the leading and lagging strands at the DNA replication fork. On the leading strand, PCNA associates with DNA polymerase and promotes continuous synthesis of long DNA strands in a processive manner. Thus, one PCNA clamp is needed per initiation event. In contrast to this, during lagging-strand synthesis, DNA replication occurs by the discontinuous production of Okazaki fragments, a process that involves a PCNA-dependent polymerase switching mechanism. In this case, one clamp needs to be loaded for every Okazaki fragment or approximately every 100-200 bp. On both strands, PCNA is loaded by RFC at a primer-template DNA junction that is later recognized by the DNA polymerase. When the polymerase completes DNA synthesis, it dissociates from the DNA and leaves the ring-shaped clamp topologically linked to the duplex, replicated DNA. This implies that the rate-limiting step for removal of PCNA from the replicated DNA (and hence loss of the replication 'imprint') is the ATP-dependent unloading of PCNA by RFC. Thus, RFC has the potential to regulate the duration of the PCNA marking on replicated DNA. This might occur in a locus-specific manner in the genome, providing a mechanism for gene-specific modulation of chromatin structure (Shibahara, 1999).

It is suggested that CAF-1-mediated nucleosome assembly normally occurs on both the leading and lagging strands immediately after passage of the DNA replication fork, as occurs in vitro when CAF-1 is present during DNA replication. Indeed, CAF-1 localizes to the sites of DNA replication in cells. But the data described here suggest that an alternative, postreplicative mechanism may also operate that could provide opportunities for asymmetric inheritance of chromatin states. If the PCNA that is used for Okazaki fragment synthesis were to remain associated with the lagging-strand product for some time after DNA replication, the amount of PCNA bound to the two sister chromatids would be inherently asymmetric. Since CAF-1 binding to PCNA can allow chromatin assembly after DNA replication, this situation would offer considerable opportunities for the establishment of an asymmetric chromatin structure on the two sister chromatids prior to the division of proliferating stem cells. Such chromatin complexes, if transferred into the daughter cells, could provide the foundation for phenotypic asymmetry of sister cells during development (Shibahara, 1999).

Although chromatin states must normally be inherited by both daughter cells, it has long been suspected that DNA replication provides a window of opportunity for changes in chromatin structures that might affect gene expression. Indeed, replication-coupled chromatin assembly suppresses basal transcription. Recent studies in the budding yeast Saccharomyces cerevisiae have showen that CAF-1 is required for the stable inheritance of transcriptionally repressed chromatin structures at telomeres and HM loci. In the absence of CAF-1, the expression of genes near the telomeres is variegated in a population of cells. It is suggested that the recruitment of CAF-1 by PCNA is required for suppression of this form of position effect variegation. Support for a role for PCNA in suppression of position effect variegation comes from genetic studies in Drosophila. A mutant in Drosophila PCNA, mus209, is a suppressor of position effect variegation and exhibits DNA repair defects that overlap with the repair defects seen in yeast lacking CAF-1. The mus209 mutant also causes strong enhancement of homeotic transformation in a trans-heterozygous crm;mus209 mutant (Yamamoto, 1997 ). The cramped (crm) gene is a Polycomb-group (Pc-G) gene, and some crm mutants show suppression of position effect variegation. PCNA and the CRM protein colocalize in nuclei of proliferating cells. These phenotypes may reflect the role of PCNA in promoting efficient coupling between DNA replication and chromatin assembly (Shibahara, 1999 and references).

The inheritance of epigenetically determined states in budding and fission yeasts may also occur by similar mechanisms. In the fission yeast S. pombe, replication proteins such as DNA polymerase, chromatin proteins Swi6 and Clr4, and histone deacetylases are involved in the epigenetic inheritance of the mating type silent chromatin and centromeric heterochromatin. It is possible that PCNA and CAF-1 mediate these effects. In the budding yeast S. cerevisiae, position effect variegation occurs at telomeres, and the establishment of chromatin repression at the mating type loci requires passage through S phase. Furthermore, heterochromatin at the mating type loci (HM) is uniquely acetylated at lysine-12 of histone H4, and a variety of mutations in the acetylated lysines of histones H3 and H4 result in defects in silencing at HM loci and telomeric reporter genes. Since CAF-1 associates with specific forms of acetylated histones, the PCNA-CAF-1-linked chromatin assembly may be involved in assembly of heterochromatin and epigenetically determined chromosomal states. PCNA interacts with the DNA-(cytosine-5)-methyltransferase at sites of DNA replication in mammalian cells, and this interaction likely mediates the inheritance of DNA methylation patterns. It is suggested that the PCNA molecules that remain associated with the replicated DNA might provide a common platform for both chromatin assembly and for DNA methylation. In some situations, this could allow preferential methylation of one sister chromatid over the other, as has been proposed for chromatin assembly (Shibahara, 1999 and references).

Formation of a heterochromatin-like structure results in transcriptional silencing at the HM mating-type loci and telomeres in Saccharomyces cerevisiae. Once formed, such epigenetically determined structures are inherited for many mitotic divisions. PCNA is important in many aspects of DNA metabolism, including DNA replication and DNA repair in eukaryotes. Human PCNA interacts with CAF-1, a three-subunit protein, to couple DNA replication or DNA repair to nucleosome deposition. In yeast, deletion of any genes encoding CAF-1 subunits (CAC1, 2 and 3) results in partial loss of silencing at telomeres and silent mating-type loci. Furthermore, mutations in the Drosophila PCNA gene mus209 suppress position effect variegation. Therefore tests were performed to see whether mutations in yeast S. cerevisiae PCNA affect silencing. Mutations in the proliferating cell nuclear antigen (PCNA), an essential component at the DNA replication fork, reduce repression of genes near a telomere and at the silent mating-type locus, HMR. The pol30-8 mutant displays coexistence of both repressed and de-repressed cells within a single colony when assayed with the ADE2 gene inserted at HMR. Unlike pol30-8, the pol30-6 and pol30-79 mutants partially reduce gene silencing at telomeres and the HMR and synergistically decreased silencing in cells lacking chromatin assembly factor 1 (CAF-1). All silencing defective mutants show reduced binding to CAF-1 in vitro and altered chromatin association of the CAF-1 large subunit in vivo. Thus two classes of pol30Delta mutants have been discovered, on the basis of their combined effect with cac1 on TPE and HMR silencing, their effect on HMR silencing, and their location on the three-dimensional structure of PCNA. Genetic analysis suggests that PCNA is involved in silencing CAF-1, both dependently and independently, providing direct mechanisms to link inheritance of DNA to the propagation of epigenetic states of gene expression in eukaryotes, a process believed to determine cell fate in complex organisms. Thus, PCNA participates in inheritance of both DNA and epigenetic chromatin structures during the S phase of the cell cycle, the latter by at least two mechanisms (Zhang, 2000).

Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p (see Drosophila Asf1) and chromatin assembly factor I (CAF-I, a heterotrimeric protein complex encoded by the CAC1-3 genes). CAF-I performs the first step of nucleosome formation, deposition of the histone (H3/H4)2 tetramer onto DNA. CAF-I delivers histones to DNA molecules that have been recently replicated either bidirectionally or during DNA repair; it is targeted to replicated DNA via a direct interaction with proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity factor. Both cacDelta asf1Delta and cacDelta hirDelta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. Biochemical and genetic studies of yeast Asf1p have revealed links to Hir protein function. In vitro, an active histone deposition complex is formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generates micrococcal nuclease-resistant DNA in the absence of DNA replication and stimulates nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p binds to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contribute to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminates telomeric gene silencing in combination with pol30-8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevent Asf1/Hir proteins from contributing to silencing. It is concluded that yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity (Sharp, 2001).

Protein interactions of the small subunit of CAF-1

Continued: see Chromatin assembly factor 1 subunit Evolutionary Homologs part 2/3 | part 3/3

Chromatin assembly factor 1 subunit: Biological Overview | Regulation | Developmental Biology | Effects of RNAi | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.