Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci

Abstract

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. In vivo, cac1/rlf2 mutants are defective in telomeric silencing and mislocalize Rap1p, a telomere-binding protein. Here, we report that in cells lacking CAF-I the silent mating loci are derepressed partially. MATa cac1 cells exhibit an unusual response to ␣-factor: They arrest and form mating projections (shmoos) initially, 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 HML␣ 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 HML. 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. We propose a model to explain the distinctions between the establishment and the maintenance of silent chromatin.

Chromatin assembly factor 1 ensures the stable maintenance of silent chromatin states in Arabidopsis

“…The stochastic nature of the TGS defects in fas mutants are distinct from other TGS mutants in which TGS is uniformly released in all cells. These observations are reminiscent of previous findings in cac budding yeast cells, which have deletions in genes encoding the subunits of CAF-1 and exhibit an increased rate of switching from transcriptionally inactive to transcriptionally active states for the silent genes at telomere and HML locus (Monson et al 1997;Enomoto & Berman 1998). In higher plants, loci at heterochromatin are marked and maintained by specific epigenetic modifications including DNA cytosine methylation, histone modifications, their specific localization in condensed heterochromatin, or the presence of short interfering RNA (Mette et al 2000;Morel et al 2000;Miura et al 2001;Gendrel et al 2002;Soppe et al 2002;Lippman et al 2003).…”

Alternative Mechanisms of Transcriptional Activation by Rap1p

“…As mentioned above, we suggest that this function is only fulfilled when two Rap1p molecules are bound to DNA. The link of Rap1p to chromatin remodeling machines via the Tox domain may be relevant for both transcriptional activation and repression (62,63). Yeast cells in which Rap1⌬Tox is the only source of Rap1p show high RAP1 expression, which is probably repressed by its genomic product Rap1p (4,25).…”

Histone chaperone ASF1 cooperates with the Brahma chromatin-remodelling machinery

“…Although the mechanism by which this is accomplished remains unclear, our data re-emphasize the importance of chromatin assembly factors in the formation of silenced chromatin. Because regularly spaced nucleosomal arrays are a landmark of silenced heterochromatin (Wallrath and Elgin 1995;Enomoto and Berman 1998), we believe that ASF1 contributes to silencing through its nucleosome assembly activity ). Therefore, the reduction of silencing in asf1 1 mutants may result from the disruption of the nucleosome array at heterochromatin.…”