Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I.

Two molecules each of the core histones H2A, H2B, H3, and H4 tightly associate with 146 bp of DNA to form a nucleosome, the fundamental repeat structure of chromatin (38; for a review, see reference 73). Numerous genetic and biochemical studies have shown that core histones also contribute to gene activation (6, 16) and repression (18, 35) and have direct roles in position-dependent gene silencing (for reviews, see references 12 and 37). The latter point has been most clearly demonstrated in the budding yeast Saccharomyces cerevisiae, in which genes adjacent to telomeres and the mating type genes present at the silent HM loci are subject to position-dependent transcriptional repression. Mutations within the N-terminal residues of histones H3 and H4 disrupt this gene silencing (22, 41, 49, 71). Furthermore, these same N-terminal regions make direct contact with SIR gene products required for silencing in S. cerevisiae (14). Thus, chromatin structures responsible for gene silencing include nucleosomes as important components. It is presently unknown how many proteins mediate assembly of histones on DNA in vivo or how such factors contribute to gene silencing. One factor involved in these processes is a three-subunit protein complex termed chromatin assembly factor I (CAF-I). CAF-I was initially purified from human somatic cell nuclear extracts on the basis of its ability to assemble histone octamers during DNA replication in vitro (62, 66; for reviews, see references 26 and 32). CAF-I-like activities have also been detected in extracts of Drosophila and Xenopus embryos (25). Purification of yeast CAF-I led to identification of the genes encoding the three yeast CAF-I subunits, each of which is homologous to its human counterpart (28). These genes have been termed CAC1, CAC2, and CAC3 (for chromosome assembly complex). Deletion of any of the three CAC genes results in decreased telomeric gene silencing (9, 28). cacΔ mutants display decreased stability of the transcriptionally silent state at both telomeres and the silent HML locus (8, 42). However, cacΔ mutants have no known growth defects. These data indicate that yeast CAF-I contributes to position-dependent gene silencing but is not the only cellular factor responsible for chromatin assembly. Loss of SIR2, SIR3, or SIR4 gene function in S. cerevisiae completely abolishes gene silencing at both HM loci and telomeres (1, 55). These genes encode proteins that are structural components of silenced heterochromatin (15, 67) and are not known to be involved in the assembly of histones on DNA. Indeed, mutants specifically defective in histone deposition have not been described. There could be several reasons for this. Formation of nucleosomes from newly synthesized histones during DNA replication is an essential process because progression through S phase of the cell cycle in the absence of histone H2B or H4 synthesis causes lethality (13, 30). However, if the process of chromatin formation were performed by multiple, partially redundant factors, recovery of mutations in this process would be difficult in standard genetic screens because of the weak phenotypes of single mutants. In this case, disruption of multiple factors would be required to observe strong phenotypes resulting from chromatin malformation or malfunction. We describe here a novel phenotype of cacΔ mutants: the residual gene silencing in these cells is sensitive to mutations in the HIR1, HIR2, and HIR3 genes and to changes in histone gene dosage. The three HIR-encoded proteins act to restrict transcription of three of the four histone gene pairs to the G1/S phase transition of the yeast cell cycle and to regulate the HTA1-HTB1 locus in response to altered levels of the gene products of this locus, histones H2A and H2B (43, 48, 60, 65). The HIR gene products regulate the histone HTA1-HTB1 promoter through a negative cis-acting site, although the Hir proteins themselves do not appear to directly contact DNA. hirΔ mutants display a Hir− phenotype: HTA1-HTB1 transcription is not repressed (i) outside of the G1/S phase; (ii) when cells are treated with hydroxyurea (HU), which inhibits DNA synthesis and causes accumulation of unassembled histone proteins; or (iii) when the HTA and HTB genes are present on high-copy-number plasmids. Although hirΔ mutants had no defects in gene silencing when CAF-I was intact, we observed a synergistic loss of telomeric and HM gene silencing in cacΔ hirΔ double mutants. These data suggest that pathways responsible for formation of heterochromatin in the absence of CAF-I are easily perturbed by changes in histone levels or histone stoichiometry. Furthermore, we provide evidence that the Hir proteins also have a role in cooperating with CAF-I to ensure proper cell growth and viability.