SUM1-1, a dominant suppressor of SIR mutations in Saccharomyces cerevisiae, increases transcriptional silencing at telomeres and HM mating-type loci and decreases chromosome stability.
ABSTRACT: Transcriptional silencing in the yeast Saccharomyces cerevisiae occurs at HML and HMR mating-type loci and telomeres and requires the products of the silent information regulator (SIR) genes. Recent evidence suggests that the silencer- and telomere-binding protein Rap1p initiates silencing by recruiting a complex of Sir proteins to the chromosome, where they act in some way to modify chromatin structure or accessibility. A single allele of the SUM1gene (SUM1-1) which restores silencing at HM loci in strains mutant for any of the four SIR genes was identified a number of years ago. However, conflicting genetic results and the lack of other alleles of SUM1 made it difficult to surmise the wild-type function of SUM1 or the manner in which the SUM1-1 mutation restores silencing in sir mutant strains. Here we report the cloning and characterization of the SUM1 gene and the SUM1-1 mutant allele. Our results indicate that SUM1-1 is an unusual altered-function mutation that can bypass the need for SIR function in HM silencing and increase repression at telomeres. A sum1 deletion mutation has only minor effects on silencing in SIR strains and does not restore silencing in sir mutants. In addition to its effect on transcriptional silencing, the SUM1-1 mutation (but not a sum1 deletion) increases the rate of chromosome loss and cell death. We suggest several speculative models for the action of SUM1-1 in silencing based on these and other data.
Project description:Gene silencing in budding yeast relies on the binding of the Silent Information Regulator (Sir) complex to chromatin, which is mediated by extensive interactions between the Sir proteins and nucleosomes. Sir3, a divergent member of the AAA+ ATPase-like family, contacts both the histone H4 tail and the nucleosome core. Here, we present the structure and function of the conserved C-terminal domain of Sir3, comprising 138 amino acids. This module adopts a variant winged helix-turn-helix (wH) architecture that exists as a stable homodimer in solution. Mutagenesis shows that the self-association mediated by this domain is essential for holo-Sir3 dimerization. Its loss impairs Sir3 loading onto nucleosomes in vitro and eliminates silencing at telomeres and HM loci in vivo. Replacing the Sir3 wH domain with an unrelated bacterial dimerization motif restores both HM and telomeric repression in sir3? cells. In contrast, related wH domains of archaeal and human members of the Orc1/Sir3 family are monomeric and have DNA binding activity. We speculate that a dimerization function for the wH evolved with Sir3's ability to facilitate heterochromatin formation.
Project description:The silent mating-type loci HML and HMR of Saccharomyces cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for the proteins Rap1, Abf1 and Sum1 as well as for the origin recognition complex (ORC). Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. In this study, we constructed a synthetic HML-E silencer (HML-SS ?I) that lacked this redundancy. It consisted solely of Rap1 and ORC-binding sites and the D2 element, a Sum1-binding site. All three elements were crucial for minimal HML silencing, and mutations in these elements led to a loss of Sir3 recruitment. The silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1?. Moreover, deletions of SIR1 and DOT1 lead to complete derepression of the HML-SS ?I silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing.
Project description:Sir protein spreading along chromosomes and silencing in Saccharomyces cerevisiae requires the NAD+-dependent histone deacetylase activity of Sir2p. We tested whether this requirement could be bypassed at the HM loci and telomeres in cells containing a stably expressed, but catalytically inactive mutant of Sir2p, sir2-345p, plus histone mutants that mimic the hypoacetylated state normally created by Sir2p. Sir protein spreading was rescued in sir2-345 mutants expressing histones in which key lysine residues in their N-termini had been mutated to arginine. Mating in these mutants was also partially restored upon overexpression of Sir3p. Together, these results indicate that histone hypoacetylation is sufficient for Sir protein spreading in the absence of production of 2'-O-acetyl-ADP ribose by sir2p and Sir2p's enzymatic function for silencing can be bypassed in a subset of cells in a given population. These results also provide genetic evidence for the existence of additional critical substrates of Sir2p for silencing in vivo.
Project description:In budding yeast, telomeres and the mating type (HM) loci are found in a heterochromatin-like silent structure initiated by Rap1 and extended by the interaction of Sir (Silencing Information Regulator) proteins with histones. Binding data demonstrate that both the H3 and H4 N terminal domains required for silencing in vivo interact directly with Sir3 and Sir4 in vitro. The role of H4 lysine 16 deacetylation is well established in Sir3 protein recruitment, however that of the H3 N terminal tail has remained unclear. In order to characterize the role of H3 in silent chromatin formation and compare it to H4 we have generated comprehensive high resolution genome-wide binding maps of heterochromatin proteins. We find that H4 lysine 16 deacetylation is required for the recruitment and spreading of heterochromatin proteins at all telomeres and HM loci. In contrast the H3 N terminus is required for neither recruitment nor spreading of Sir proteins. Instead, deletion of the H3 tail leads to increased accessibility within heterochromatin of an ectopic bacterial dam methylase and the decreased mobility of an HML heterochromatic fragment in sucrose gradients. These findings indicate an altered chromatin structure. We propose that Sir proteins recruited by the H4 tail then interact with the H3 tail to form a higher order silent chromatin structure. Overall design: Sir3, Sir4, and Rap1 were ChIP'd from wild type budding yeast strains and normalized to input. Sir3 and Rap1 were ChIP'd from mutant budding yeast strains and normalized to input.
Project description:Silent information regulator proteins Sir2, Sir3, and Sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (HM) loci in budding yeast. We have performed a detailed biochemical and genetic analysis of the largest Sir protein, Sir4. The N-terminal half of Sir4 is dispensable for SIR-mediated repression of HM loci in vivo, except in strains that lack Yku70 or have weak silencer elements. For HM silencing in these cells, the C-terminal domain (Sir4C, residues 747-1,358) must be complemented with an N-terminal domain (Sir4N; residues 1-270), expressed either independently or as a fusion with Sir4C. Nonetheless, recombinant Sir4C can form a complex with Sir2 and Sir3 in vitro, is catalytically active, and has sedimentation properties similar to a full-length Sir4-containing SIR complex. Sir4C-containing SIR complexes bind nucleosomal arrays and protect linker DNA from nucleolytic digestion, but less effectively than wild-type SIR complexes. Consistently, full-length Sir4 is required for the complete repression of subtelomeric genes. Supporting the notion that the Sir4 N-terminus is a regulatory domain, we find it extensively phosphorylated on cyclin-dependent kinase consensus sites, some being hyperphosphorylated during mitosis. Mutation of two major phosphoacceptor sites (S63 and S84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (P2A), which alone can enhance the stability of either the repressed or active state. The triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. We conclude that the Sir4 N-terminus plays two roles in SIR-mediated silencing: it contributes to epigenetic repression by stabilizing the SIR-mediated protection of linker DNA; and, as a target of phosphorylation, it can destabilize silencing in a regulated manner.
Project description:The S-M checkpoint ensures that entry into mitosis is dependent on completion of DNA replication. In the fission yeast Schizosaccharomyces pombe, the SM checkpoint mutant cdc2-3w is thought to be defective in receiving the checkpoint signal. To isolate genes that function in the checkpoint pathway, we screened an S. pombe cDNA library for genes that, when overexpressed, could suppress the checkpoint defect of cdc2-3w. Using this approach, we have identified a novel gene, sum1+ (suppressor of uncontrolled mitosis). sum1+ encodes a highly conserved WD-transducin repeat protein with striking sequence similarity to the human transforming growth factor (TGF)-beta-receptor interacting protein TRIP-1 and to the translation initiation factor 3 subunit eIF3-p39, encoded by the TIF34 gene in Saccharomyces cerevisiae. S. pombe sum1+ is an essential gene, required for normal cell growth and division. In addition to restoring checkpoint control, overexpression of sum1+ inhibits the normal cell cycle response to osmotic stress. Furthermore, we demonstrate that inactivation of the stress-activated MAP kinase pathway, required for cell cycle stress response, restores the S-M checkpoint in cdc2-3w cells. These results suggest that Suml interacts with the stress-activated MAP kinase pathway and raise the possibility that environmental conditions may influence the checkpoint response in fission yeast.
Project description:In budding yeast, telomeres and the mating type (HM) loci are found in a heterochromatin-like silent structure initiated by Rap1 and extended by the interaction of Silencing Information Regulator (Sir) proteins with histones. Binding data demonstrate that both the H3 and H4 N-terminal domains required for silencing in vivo interact directly with Sir3 and Sir4 in vitro. The role of H4 lysine 16 deacetylation is well established in Sir3 protein recruitment; however, that of the H3 N-terminal tail has remained unclear. To characterize the role of H3 in silent chromatin formation and compare it to H4 we have generated comprehensive high resolution genome-wide binding maps of heterochromatin proteins. We found that H4 lysine 16 deacetylation is required for the recruitment and spreading of heterochromatin proteins at all telomeres and HM loci. In contrast, the H3 N terminus is required for neither recruitment nor spreading of Sir proteins. Instead, deletion of the H3 tail leads to increased accessibility within heterochromatin of an ectopic bacterial dam methylase and the decreased mobility of an HML heterochromatic fragment in sucrose gradients. These findings indicate an altered chromatin structure. We propose that Sir proteins recruited by the H4 tail then interact with the H3 tail to form a higher order silent chromatin structure.
Project description:Cohesin is a key determinant of chromosome architecture due to its DNA binding and tethering ability. Cohesin binds near centromeres and chromosome arms and also close to telomeres, but its role near telomeres remains elusive. In budding yeast, transcription within 20 kb of telomeres is repressed, in part by the histone-modifying silent information regulator (SIR) complex. However, extensive subtelomeric repressed domains lie outside the SIR-binding region, but the mechanism of silencing in these regions remains poorly understood. Here, we report a role for cohesin in subtelomeric silencing that extends even beyond the zone of SIR binding. Clusters of subtelomeric genes were preferentially derepressed in a cohesin mutant, whereas SIR binding was unaltered. Genetic interactions with known telomere silencing factors indicate that cohesin operates independent of the SIR-mediated pathway for telomeric silencing. Mutant cells exhibited Mpk1-dependent Sir3 hyperphosphorylation that contributes to subtelomeric derepression to a limited extent. Compaction of subtelomeric domains and tethering to the nuclear envelope were impaired in mutant cells. Our findings provide evidence for a unique SIR-independent mechanism of subtelomeric repression mediated by cohesin.
Project description:Histones of heterochromatin are deacetylated in yeast and methylated in more complex eukaryotes to regulate heterochromatin structure and gene silencing. Here, we report that histone H2A phosphorylated at serine 129 (?H2A) in Saccharomyces cerevisiae is a conceptually new type of heterochromatin modification that functions downstream of silent chromatin assembly. We show that ?H2A is enriched throughout yeast telomeric and silent mating locus (HM) heterochromatin where ?H2A results from the action of kinases Tel1 and Mec1. Interestingly, mutation of ?H2A has no apparent effect on the binding of Sir (silent information regulator) complex or on gene silencing. In contrast, deletion of SIR3 abolishes the formation of ?H2A at heterochromatin. To address the function of ?H2A, we used a ?rif1 mutant strain in which telomeres are excessively elongated to show that ?H2A is required for the optimal recruitment of Cdc13, a regulator of telomere elongation, and for telomere elongation itself. Thus, a histone modification that parallels Sir3 protein binding is shown here to be dispensable for the formation of a silent structure but is important for a crucial heterochromatin-specific downstream function in telomere homeostasis.
Project description:BACKGROUND: Replication initiation at origins of replication in the yeast genome takes place on chromatin as a template, raising the question how histone modifications, for instance histone acetylation, influence origin firing. Initiation requires binding of the replication initiator, the Origin Recognition Complex (ORC), to a consensus sequence within origins. In addition, other proteins bind to recognition sites in the vicinity of ORC and support initiation. In previous work, we identified Sum1 as an origin-binding protein that contributes to efficient replication initiation. Sum1 is part of the Sum1/Rfm1/Hst1 complex that represses meiotic genes during vegetative growth via histone deacetylation by the histone deacetylase (HDAC) Hst1. RESULTS: In this study, we investigated how Sum1 affected replication initiation. We found that it functioned in initiation as a component of the Sum1/Rfm1/Hst1 complex, implying a role for histone deacetylation in origin activity. We identified several origins in the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, sum1Delta or hst1Delta caused a significant increase in histone H4 lysine 5 (H4 K5) acetylation levels, but not other H4 acetylation sites, at those origins. Furthermore, mutation of lysines to glutamines in the H4 tail, which imitates the constantly acetylated state, resulted in a reduction of origin activity comparable to that in the absence of Hst1, showing that deacetylation of H4 was important for full initiation capacity of these origins. CONCLUSION: Taken together, our results demonstrate a role for histone deacetylation in origin activity and reveal a novel aspect of origin regulation by chromatin. These results suggest recruitment of the Sum1/Rfm1/Hst1 complex to a number of yeast origins, where Hst1 deacetylated H4 K5.