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).

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).