Project description:In the budding yeast, HMR, HML, telomere and rDNA domain are known as a silencing region. Sir2 need to make it at rDNA and, HMR, HML and the telomere need to Sir2, Sir3, Sir4 complex to control internal gene repression. In this report, we found a newly Sir3 binding domain, CN domain (Chromosome New region) 1~14, by the ChIP on chip analysis on S.cerevisiae chromosome. In addition, we also performed ChIP on chip analysis with anti-Sir3 antibody using G1 phase synchronized cell to find Sir3 distribution difference of stage of cell cycle and we found CN15~CN25 which was G1 phase specific Sir3 binding region. Furthermore, we analyzed difference of gene expression at CN region in sir3 strain, and some regions did not change level of gene expression. In the conventional report, Sir3 had recruited by Sir2 and Sir4 on chromosome, but recruit of Sir3 was independent on Sir2 and Sir4 at some CN regions. These data suggested that we found a newly Sir3 function and Sir3 recruited system on chromosome.
Project description:O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. chromatin affinity-precipitation (ChAP) method was used to detect the chromatin fragments at which small molecules interact with binding partners. Chromatin immunoprecipitation of Sir3 and of Sir2, respectively, applied with tilling array chip (ChIP on chip of Sir3 and of Sir2, respectively) and Chromatin affinity-precipitation of AAR applied with tilling array chip (ChAP on chip of AAR ) analysis demonstrated that an extended spreading of Sir3 and of AAR, but not Sir2 in Saccharomyces cerevisiae Ysa1 deleted cells compared with those in wild type cells
Project description:O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. chromatin affinity-precipitation (ChAP) method was used to detect the chromatin fragments at which small molecules interact with binding partners. Chromatin immunoprecipitation of Sir3 and of Sir2, respectively, applied with tilling array chip (ChIP on chip of Sir3 and of Sir2, respectively) and Chromatin affinity-precipitation of AAR applied with tilling array chip (ChAP on chip of AAR ) analysis demonstrated that an extended spreading of Sir3 and of AAR, but not Sir2 in Saccharomyces cerevisiae Ysa1 deleted cells compared with those in wild type cells Comparison the distributions of Sir3, of Sir2 and of AAR on silent heterochromatin of Ysa1 deletion cells vs those of wild type cells
Project description:Gene silencing at the mating type loci in budding yeast depends on the Sir proteins. Sir2, Sir3 and Sir4 are indispensable for silencing, whereas Sir1 has a more limited role. The Sir proteins are also involved in repression at telomeres and ribosomal DNA (rDNA) repeats. Proposed mechanisms for Sir-mediated silencing include limiting access to silenced DNA and inhibition of transcript initiation and/or elongation. Using an inducible M.SssI DNA methyltransferase expression system, we showed previously that the silenced mating type loci are methylated at a much slower rate than the rest of the genome in vivo. Here, we show that, in the absence of Sir2, Sir3 or Sir4, the silenced loci and the telomeric X elements are methylated at a similar rate to the rest of the genome, indicating that these Sir proteins impede access, but do not prevent it. Loss of Sir1 has a minor effect. The rDNA repeats are methylated faster in the absence of Sir2 and, to a lesser extent, of Sir3, but not of Sir4. Our data indicate that steric occlusion is unlikely to be the primary mechanism of silencing, since silenced DNA is accessible in vivo, albeit at a slower rate than elsewhere in the genome.
Project description:Sir2 is a highly conserved NAD+-dependent histone deacetylase that functions in heterochromatin formation and promotes replicative lifespan (RLS) in the budding yeast, Saccharomyces cerevisiae. Within the yeast rDNA locus, Sir2 is required for efficient cohesin recruitment and maintaining stability of the tandem array. In addition to the previously reported depletion of Sir2 in replicatively aged cells, we discovered that subunits of the Sir2 containing complexes, SIR and RENT, were depleted. Several other rDNA structural protein complexes also exhibited age-related depletion, most notably the cohesin complex. We hypothesized that mitotic chromosome instability (CIN) due to cohesin depletion could be a driver of replicative aging. ChIP assays of the residual cohesin (Mcd1-13xMyc) in moderately aged cells showed strong depletion from the rDNA and initial redistribution to the point centromeres, which was then lost in older cells. Despite the shift in cohesin distribution, sister chromatid cohesion was partially attenuated in aged cells and the frequency of chromosome loss was increased. This age-induced CIN was exacerbated in strains lacking Sir2 and its paralog, Hst1, but suppressed in strains that stabilize the rDNA array due to deletion of FOB1 or through caloric restriction (CR). Furthermore, ectopic expression of MCD1 from a doxycycline-inducible promoter was sufficient to suppress rDNA instability in aged cells and to extend RLS. Taken together we conclude that age-induced depletion of cohesin and multiple other nucleolar chromatin factors destabilize the rDNA locus, which then results in general CIN and aneuploidy that shortens RLS.
Project description:As part of a study of establishment of silencing in Saccharomyces cerevisiae, we performed ChIP-seq on myc-tagged Sir4 in several conditions. Included in those conditions are wild-type cycling cells, cycling sir3∆ cells, and various experiments during which silencing establishment was controlled using the inducible SIR3-EBD allele. Silencing establishment experiments were performed in both wild-type and dot1∆ cells.
Project description:Telomere chromatin structure is pivotal for maintaining genome stability by regulating the binding of telomere-associated proteins and inhibition of a DNA damage response. In yeast, the silent information regulator (Sir) proteins bind to terminal telomeric repeats and to subtelomeric X-elements resulting in histone deacetylation and transcriptional silencing. Herein, we show that sir2 mutant strains display a very specific loss of a nucleosome residing in the X-element. Most yeast telomeres contain an X-element and the nucleosome occupancy defect in sir2 mutants is remarkably consistent between different telomeres.