Depletion of Limiting rDNA Structural Complexes Triggers Chromosomal Instability and Replicative Aging of Saccharomyces cerevisiae.
ABSTRACT: Sir2 is a highly conserved NAD+-dependent histone deacetylase that functions in heterochromatin formation and promotes replicative life span (RLS) in the budding yeast, Saccharomyces cerevisiae Within the yeast rDNA locus, Sir2 is required for efficient cohesin recruitment and maintaining the 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 silent information regulator (SIR) and regulator of nucleolar silencing and telophase (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. Chromatin immunoprecipitation assays of the residual cohesin (Mcd1-Myc) 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. 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: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:In Saccharomyces cerevisiae, replicative life span (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD<sup>+</sup>-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1Δ mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1Δ cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.
Project description:The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions. We show further that long-range Fob1-mediated Ter-Ter interactions in trans are downregulated by Sir2. These regulatory mechanisms control intrachromatid recombination and the replicative life span (RLS).
Project description:Activation of Sir2 orthologs is proposed to increase lifespan downstream of dietary restriction. Here, we describe an examination of the effect of 32 different lifespan-extending mutations and four methods of DR on replicative lifespan (RLS) in the short-lived sir2? yeast strain. In every case, deletion of SIR2 prevented RLS extension; however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2? lifespan defect is a rare phenotype among longevity interventions and suggest that sir2? cells senesce rapidly by a mechanism distinct from that of wild-type cells. They also demonstrate that failure to observe lifespan extension in a short-lived background, such as cells or animals lacking sirtuins, should be interpreted with caution.
Project description:The fungal pathogen, Cryptococcus neoformans, has been shown to undergo replicative aging. Old cells are characterized by advanced generational age and phenotypic changes that appear to mediate enhanced resistance to host and antifungal-based killing. As a consequence of this age-associated resilience, old cells accumulate during chronic infection. Based on these findings, we hypothesized that shifting the generational age of a pathogenic yeast population would alter its vulnerability to the host and affect its virulence. SIR2 is a well-conserved histone deacetylase, and a pivotal target for the development of anti-aging drugs. We tested its effect on C. neoformans' replicative lifespan (RLS). First, a mutant C. neoformans strain (sir2?) was generated, and confirmed a predicted shortened RLS in sir2? cells consistent with its known role in aging. Next, RLS analysis showed that treatment of C. neoformans with Sir2p-agonists resulted in a significantly prolonged RLS, whereas treatment with a Sir2p-antagonist shortened RLS. RLS modulating effects were dependent on SIR2 and not observed in sir2? cells. Because SIR2 loss resulted in a slightly impaired fitness, effects of genetic RLS modulation on virulence could not be compared with wild type cells. Instead we chose to chemically modulate RLS, and investigated the effect of Sir2p modulating drugs on C. neoformans cells in a Galleria mellonella infection model. Consistent with our hypothesis that shifts in the generational age of the infecting yeast population alters its vulnerability to host cells, we observed decreased virulence of C. neoformans in the Galleria host when RLS was prolonged by treatment with Sir2p agonists. In contrast, treatment with a Sir2p antagonist, which shortens RLS enhanced virulence in Galleria. In addition, combination of Sir2p agonists with antifungal therapy enhanced the antifungal's effect. Importantly, no difference in virulence was observed with drug treatment when sir2? cells were used for infection, which confirmed target specificity and ruled out non-specific effects of the drugs on the Galleria host. Thus, this study suggests that RLS modulating drugs, such as Sir2p agonists, shift lifespan and vulnerability of the fungal population, and should be further investigated as a potential class of novel antifungal drug targets that can enhance antifungal efficacy.
Project description:Sir2 and the homologous proteins, Hst1, Hst2, Hst3, and Hst4 from Saccharomyces cerevisiae are NAD+-dependent histone deacetylases of the sirtuin protein family. Sir2 functions in transcriptional silencing at the silent mating-type loci, telomeres, and rDNA locus, but also promotes replicative lifespan. To gain a better understanding of the chromatin-regulatory roles carried out by Sir2 and the Hst proteins, we performed ChIP-sequencing analysis on all five sirtuins and Sum1, the DNA binding partner for Hst1. Sir2, Hst1, and Sum1 were abundantly, and functionally co-enriched at several major targets, including the telomeric repeats, where they were required for maintaining proper telomere repeat length. At tRNA target genes they were required for efficient cohesin and condensin deposition. Across the open reading frames of glycolytic and ribosomal protein genes, Sir2 and Hst1 functioned in NAD+-dependent transcriptional repression at the diauxic shift, directly linking Sir2 to glucose metabolism, which could have implications for longevity. Six factors and Input ChIP-seq samples were analyzed in Saccharomyces eerevisiae.
Project description:The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus.
Project description:Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.
Project description:Silent information regulator 2 (Sir2), the founding member of the conserved sirtuin family of NAD(+)-dependent histone deacetylase, regulates several physiological processes including genome stability, gene silencing, metabolism and life span in yeast. Within the nucleus, Sir2 is associated with telomere clusters in the nuclear periphery and rDNA in the nucleolus and regulates gene silencing at these genomic sites. How distribution of Sir2 between telomere and rDNA is regulated is not known. Here we show that Sir2 is sumoylated and this modification modulates the intra-nuclear distribution of Sir2. We identify Siz2 as the key SUMO ligase and show that multiple lysines in Sir2 are subject to this sumoylation activity. Mutating K215 alone counteracts the inhibitory effect of Siz2 on telomeric silencing. SUMO modification of Sir2 impairs interaction with Sir4 but not Net1 and, furthermore, SUMO modified Sir2 shows predominant nucleolar localization. Our findings demonstrate that sumoylation of Sir2 modulates distribution between telomeres and rDNA and this is likely to have implications for Sir2 function in other loci as well.
Project description:The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized SCC3 to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.