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.     

Identification of a novel allele of SIR3 defective in the maintenance, but not the establishment, of silencing in Saccharomyces cerevisiae

Using a screen for genes that affect telomere function, we isolated sir3-P898R, an allele of SIR3 that reduces telomeric silencing yet does not affect mating. While sir3-P898R mutations cause no detectable mating defect in quantitative assays, they result in synergistic mating defects in combination with mutations such as sir1 that affect the establishment of silencing. In contrast, sir3-P898R in combination with a cac1 mutation, which affects the maintenance of silencing, does not result in synergistic mating defects. MATa sir3-P898R mutants form shmoo clusters in response to alpha-factor, and sir3-P898R strains are capable of establishing silencing at a previously derepressed HML locus with kinetics like that of wild-type SIR3 strains. These results imply that Sir3-P898Rp is defective in the maintenance, but not the establishment of silencing. In addition, overexpression of a C-terminal fragment of Sir3-P898R results in a dominant nonmating phenotype: HM silencing is completely lost at both HML and HMR. Furthermore, HM silencing is most vulnerable to disruption by the Sir3-P898R C terminus immediately after S-phase, the time when new silent chromatin is assembled onto newly replicated DNA.     

SUM1, an Apparent Positive Regulator of the Cryptic Mating-Type Loci in SACCHAROMYCES CEREVISIAE

The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci. To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation. A strain with the genotype HMLa MAT alpha HMRa mar1-1 is unable to mate because of the simultaneous expression of a and alpha information. A mutant of this strain was isolated that exhibits an alpha phenotype and, therefore, presumably fails to express the HML and HMR loci. We designate the new locus SUM1 (suppressor of mar). The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci. The sum1 mutation affects expression of both a and alpha information at the HM loci. Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4. The sum1-1 mutation has no discernible phenotype in a Mar+ strain. We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.     

Locus specificity determinants in the multifunctional yeast silencing protein Sir2

Yeast SIR2, the founding member of a conserved gene family, acts to modulate chromatin structure in three different contexts: silent (HM) mating-type loci, telomeres and rDNA. At HM loci and telomeres, Sir2p forms a complex with Sir3p and Sir4p. However, Sir2p's role in rDNA silencing is Sir3/4 independent, requiring instead an essential nucleolar protein, Net1p. We describe two novel classes of SIR2 mutations specific to either HM/telomere or rDNA silencing. Despite their opposite effects, both classes of mutations cluster in the same two regions of Sir2p, each of which borders on a conserved core domain. A surprising number of these mutations are dominant. Several rDNA silencing mutants display a Sir2p nucleolar localization defect that correlates with reduced Net1p binding. Although the molecular defect in HM/telomere-specific mutants is unclear, they mimic an age-related phenotype where Sir3p and Sir4p relocalize to the nucleolus. Artificial targeting can circumvent the silencing defect in a subset of mutants from both classes. These results define distinct functional domains of Sir2p and provide evidence for additional Sir2p-interacting factors with locus-specific silencing functions.     

Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, silencing at the HM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region at HMR. Sir1p, in contrast, associated only with the HMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading across HMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states.     

Efficient production of a ring derivative of chromosome III by the mating-type switching mechanism in Saccharomyces cerevisiae.

The mating-type switches in the yeast Saccharomyces cerevisiae occur by unidirectional transposition of replicas of unexpressed genetic information, residing at HML or HMR, into the mating-type locus (MAT). The source loci, HML and HMR, remain unchanged. Interestingly, when the HM cassettes are expressed, as in marl strains, the HML and HMR cassettes can also efficiently switch, apparently by obtaining genetic information from either of the other two cassettes (Klar et al., Cell 25:517-524, 1981). We have isolated a novel chromosome III rearrangement in heterothallic (marl ho) strains, which is also produced efficiently in marl HO cells, presumably the consequence of a recombination event between HML and HMR. The fusion results in the loss of sequences which are located distal to HML and to HMR and produces a ring derivative of chromosome III. Cells containing such a ring chromosome are viable as haploids; apparently, no essential loci are located distal to the HM loci. The fusion cassette behaves as a standard HM locus with respect to both regulation by the MAR/SIR control and its role in switching MAT.