Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae.
ABSTRACT: The nucleolus is speculated to be a regulator of cellular senescence in numerous biological systems (Guarente, Genes Dev 11(19):2449-2455, 1997; Johnson et al., Curr Opin Cell Biol 10(3):332-338, 1998). In the budding yeast Saccharomyces cerevisiae, alterations in nucleolar architecture, the redistribution of nucleolar protein and the accumulation of extrachromosomal ribosomal DNA circles (ERCs) during replicative aging have been reported. However, little is known regarding rDNA stability and changes in nucleolar activity during chronological aging (CA), which is another yeast aging model used. In the present study, the impact of aberrant cell cycle checkpoint control (knock-out of BUB1, BUB2, MAD1 and TEL1 genes in haploid and diploid hemizygous states) on CA-mediated changes in the nucleolus was studied. Nucleolus fragmentation, changes in the nucleolus size and the nucleolus/nucleus ratio, ERC accumulation, expression pattern changes and the relocation of protein involved in transcriptional silencing during CA were revealed. All strains examined were affected by oxidative stress, aneuploidy (numerical rather than structural aberrations) and DNA damage. However, the bub1 cells were the most prone to aneuploidy events, which may contribute to observed decrease in chronological lifespan. We postulate that chronological aging may be affected by redox imbalance-mediated chromosome XII instability leading to both rDNA instability and whole chromosome aneuploidy. CA-mediated nucleolus fragmentation may be a consequence of nucleolus enlargement and/or Nop2p upregulation. Moreover, the rDNA content of chronologically aging cells may be a factor determining the subsequent replicative lifespan. Taken together, we demonstrated that the nucleolus state is also affected during CA in yeast.
Project description: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:The nucleolus is a membraneless organelle of the nucleus and the site of rRNA synthesis, maturation, and assembly into preribosomal particles. The nucleolus, organized around arrays of rRNA genes (rDNA), dissolves during prophase of mitosis in metazoans, when rDNA transcription ceases, and reforms in telophase, when rDNA transcription resumes. No such dissolution and reformation cycle exists in budding yeast, and the precise course of nucleolar segregation remains unclear. By quantitative live-cell imaging, we observed that the yeast nucleolus is reorganized in its protein composition during mitosis. Daughter cells received equal shares of preinitiation factors, which bind the RNA polymerase I promoter and the rDNA binding barrier protein Fob1, but only about one-third of RNA polymerase I and the processing factors Nop56 and Nsr1. The distribution bias was diminished in nonpolar chromosome segregation events observable in dyn1 mutants. Unequal distribution, however, was enhanced by defects in RNA polymerase I, suggesting that rDNA transcription supports nucleolar segregation. Indeed, quantification of pre-rRNA levels indicated ongoing rDNA transcription in yeast mitosis. These data, together with photobleaching experiments to measure nucleolar protein dynamics in anaphase, consolidate a model that explains the differential partitioning of nucleolar components in budding yeast mitosis.
Project description:Nutrient starvation or inactivation of target of rapamycin complex 1 (TORC1) in budding yeast induces nucleophagy, a selective autophagy process that preferentially degrades nucleolar components. DNA, including ribosomal DNA (rDNA), is not degraded by nucleophagy, even though rDNA is embedded in the nucleolus. Here, we show that TORC1 inactivation promotes relocalization of nucleolar proteins and rDNA to different sites. Nucleolar proteins move to sites proximal to the nuclear-vacuolar junction (NVJ), where micronucleophagy (or piecemeal microautophagy of the nucleus) occurs, whereas rDNA dissociates from nucleolar proteins and moves to sites distal to NVJs. CLIP and cohibin, which tether rDNA to the inner nuclear membrane, were required for repositioning of nucleolar proteins and rDNA, as well as effective nucleophagic degradation of the nucleolar proteins. Furthermore, micronucleophagy itself was necessary for the repositioning of rDNA and nucleolar proteins. However, rDNA escaped from nucleophagic degradation in CLIP- or cohibin-deficient cells. This study reveals that rDNA-nucleolar protein separation is important for the nucleophagic degradation of nucleolar proteins.
Project description:Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.
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. Overall design: 2 WT HiC samples with 2 sequencing runs each.
Project description:The ribosomal DNA (rDNA) is the most evolutionarily conserved segment of the genome and gives origin to the nucleolus, an energy intensive nuclear organelle and major hub influencing myriad molecular processes from cellular metabolism to epigenetic states of the genome. The rDNA/nucleolus has been directly and mechanistically implicated in aging and longevity in organisms as diverse as yeasts, Drosophila, and humans. The rDNA is also a significant target of DNA methylation that silences supernumerary rDNA units and regulates nucleolar activity. Here, we introduce an age clock built exclusively with CpG methylation within the rDNA. The ribosomal clock is sufficient to accurately estimate individual age within species, is responsive to genetic and environmental interventions that modulate life-span, and operates across species as distant as humans, mice, and dogs. Further analyses revealed a significant excess of age-associated hypermethylation in the rDNA relative to other segments of the genome, and which forms the basis of the rDNA clock. Our observations identified an evolutionarily conserved marker of aging that is easily ascertained, grounded on nucleolar biology, and could serve as a universal marker to gauge individual age and response to interventions in humans as well as laboratory and wild organisms across a wide diversity of species.
Project description:During chronological aging of budding yeast cells, the culture medium can become acidified, and this acidification limits cell survival. As a consequence, buffering the culture medium to pH 6 significantly extends chronological life span under standard conditions in synthetic medium. In this study, we assessed whether a similar process occurs during replicative aging of yeast cells. We find no evidence that buffering the pH of the culture medium to pH levels either higher or lower than the initial pH of the medium is able to significantly extend replicative lifespan. Thus, we conclude that, unlike chronological life span, replicative life span is not limited by acidification of the culture medium or by changes in the pH of the environment.
Project description:Dynamic changes in gene positioning contribute to differential expression of virulence-related gene families in protozoan pathogens; however, the role of nuclear architecture in gene expression in the human malaria parasite Plasmodium falciparum remains poorly understood. Here we investigated the developmentally regulated ribosomal RNA (rRNA) gene family in P. falciparum, which, unlike that in most eukaryotes, contains only a few unlinked copies of rRNA genes scattered over the subtelomeric regions of several chromosomes. We show that active and silent members of this gene family cluster in a single perinuclear nucleolus. This rDNA nuclear confinement is DNA sequence dependent, as plasmids carrying rDNA fragments are targeted to the nucleolus. Likewise, insertion of an rDNA sequence into a subtelomere from a chromosome lacking rRNA genes leads to repositioning in the nucleolus. Furthermore, we observed that rDNA spatial organization restricted interchromosomal interactions, as chromosome end-bearing rRNA genes were found to be preferentially juxtaposed, demonstrating nonrandom association of telomeres. Using Br-UTP incorporation, we observed two alpha-amanitin-resistant nucleolar transcription sites that disappeared when the rDNA cluster broke up in the replicative blood stages. Taken together, our results provide conceptual insights into functionally differentiated nuclear territories and their role in gene expression in malaria parasites.
Project description:Acs2p is one of two acetyl-coenzyme A synthetases in Saccharomyces cerevisiae. We have prepared and characterized a monoclonal antibody specific for Acs2p and find that Acs2p is localized primarily to the nucleus, including the nucleolus, with a minor amount in the cytosol. We find that Acs2p is required for replicative longevity: an acs2 Delta strain has a reduced replicative life span compared to wild-type and acs1 Delta strains. Furthermore, replicatively aged acs2 Delta cells contain elevated levels of extrachromosomal rDNA circles, and silencing at the rDNA locus is impaired in an acs2 Delta strain. These findings indicate that Acs2p-mediated synthesis of acetyl-CoA in the nucleus functions to promote rDNA silencing and replicative longevity in yeast.
Project description:The nucleolus is a subnuclear compartment with key roles in rRNA synthesis and ribosome biogenesis, complex processes that require hundreds of proteins and factors. Alterations in nucleolar morphology and protein content have been linked to the control of cell proliferation and stress responses and, recently, further implicated in cell senescence and ageing. In this study, we report the functional role of NOL12 in the nucleolar homeostasis of human primary fibroblasts. NOL12 repression induces specific changes in nucleolar morphology, with increased nucleolar area but reduced nucleolar number, along with nucleolar accumulation and increased levels of fibrillarin and nucleolin. Moreover, NOL12 repression leads to stabilization and activation of p53 in an RPL11-dependent manner, which arrests cells at G2 phase and ultimately leads to senescence. Importantly, we found NOL12 repression in association with nucleolar stress-like responses in human fibroblasts from elderly donors, disclosing it as a biomarker in human chronological aging.