The Role of Sas2, an Acetyltransferase Homologue of Saccharomyces Cerevisiae, in Silencing and Orc Function

Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2Δ) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2Δ required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2Δ suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2Δ had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed.     

A role for a replicator dominance mechanism in silencing.

The role of the natural HMR-E silencer in modulating replication initiation and silencing by the origin recognition complex (ORC) was examined. When natural HMR-E was the only silencer controlling HMR, the silencer's ORC-binding site (ACS) was dispensable for replication initiation but essential for silencing, indicating that a non-silencer chromosomal replicator(s) existed in close proximity to the silencer. Further analysis revealed that regions flanking both sides of HMR-E contained replicators. In contrast to replication initiation by the intact silencer, initiation by the non-silencer replicator(s) was abolished in an orc2-1 mutant, indicating that these replicators were extremely sensitive to defects in ORC. Remarkably, the activity of one of the non-silencer replicators correlated with reduced silencing; inactivation of these replicators caused by either the orc2-1 mutation or the deletion of flanking sequences enhanced silencing. These data were consistent with a role for the ORC bound to the HMR-E silencer ACS in suppressing the function of neighboring ORC molecules capable of inhibiting silencing, and indicated that differences in ORC-binding sites within HMR itself had profound effects on ORC function. Moreover, replication initiation by natural HMR-E was inefficient, suggesting that closely spaced replicators within HMR contributed to an inhibition of replication initiation.     

Tolerance of Sir1p/origin recognition complex-dependent silencing for enhanced origin firing at HMRa

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.     

A novel role for histone chaperones CAF-1 and Rtt106p in heterochromatin silencing

The histone chaperones CAF-1 and Rtt106p are required for heterochromatin silencing in the yeast Saccharomyces cerevisiae. Although it has been suggested that CAF-1 is involved in the maintenance of heterochromatin silencing, their exact functions during this process are not well understood. Here, we show that CAF-1 and Rtt106p are involved in the early stages of heterochromatin formation. The binding of Sir proteins to telomeric heterochromatin is significantly reduced and, additionally, Sir proteins are mislocalized in cells lacking CAF-1 and Rtt106p. At the HMR locus, CAF-1 and Rtt106p are required for the initial recruitment of Sir2p and Sir3p, but not Sir4p, to the HMR-E silencer, where silencing initiates, as well as the efficient spreading of all of these Sir proteins to the distal a1 gene. Moreover, silencing at the HMR locus is dramatically reduced in cells lacking CAF-1, Rtt106p, and Sir1p. Thus, these studies reveal a novel role for CAF-1 and Rtt106p in epigenetic silencing and indicate that the spreading of heterochromatin, a poorly understood process, requires histone chaperones.     

Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site

Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.     

Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.     

A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.