RESUMEN
Genome instability is a cause of cellular senescence. The ribosomal RNA gene repeat (rDNA) is one of the most unstable regions in the genome and its instability is proposed to be a major inducer of cellular senescence and restricted lifespan. We previously conducted a genome-wide screen using a budding yeast deletion library to identify mutants that exhibit a change in the stability of the rDNA region, compared to the wild-type. To investigate the correlation between rDNA stability and lifespan, we examined deletion mutants with very stable rDNA and found that deletion of EAF3, encoding a component of the NuA4 histone acetyltransferase complex, reproducibly resulted in increased stabilization of the rDNA. In the absence of Eaf3, and of other subunits of the NuA4 complex, we observed lower levels of extrachromosomal rDNA circles that are produced by recombination in the rDNA and are thus an indicator of rDNA instability. The replicative lifespan in the eaf3 mutant was extended by ~30%, compared to the wild-type strain. Our findings provide evidence that rDNA stability is correlated with extended replicative lifespan. The eaf3 mutation possibly affects the non-coding transcription in rDNA that regulates rDNA recombination through cohesin dissociation.
Asunto(s)
Senescencia Celular/genética , ADN Ribosómico/fisiología , Genes de ARNr , Histona Acetiltransferasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Acetiltransferasas/genética , Acetiltransferasas/fisiología , Proteínas de Unión al ADN/fisiología , Regulación Fúngica de la Expresión Génica , Inestabilidad Genómica , Histona Acetiltransferasas/genética , Mutación , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Telómero/fisiología , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/fisiología , Silenciador del Gen/fisiología , Histonas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Telómero/genética , Telómero/metabolismo , CohesinasRESUMEN
We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles of SIR and RNA interference (RNAi) genes in T. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography of chromatin at the HML and HMR loci than was observed in S. cerevisiae. S. cerevisiae Sir1, enriched at the silencers of HMLα and HMR A: , was absent from telomeres and did not repress subtelomeric genes. In contrast to S. cerevisiae SIR1's partially dispensable role in silencing, the T. delbrueckii SIR1 paralog KOS3 was essential for silencing. KOS3 was also found at telomeres with T. delbrueckii Sir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing, T. delbrueckii RNAi genes AGO1 and DCR1 did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.
Asunto(s)
Proteínas Fúngicas/genética , Silenciador del Gen , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Torulaspora/genética , Sitios de Unión , Centrómero , Mapeo Cromosómico , Cromosomas Fúngicos , Evolución Molecular , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Telómero , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Changes in the locations and boundaries of heterochromatin are critical during development, and de novo assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. De novo assembly cannot occur in the G1 phase of the cell cycle and one to two divisions are needed for complete silent chromatin assembly and transcriptional repression. Mutation of DOT1, the histone H3 lysine 79 (K79) methyltransferase, and SET1, the histone H3 lysine 4 (K4) methyltransferase, speed de novo assembly. These observations have led to the model that regulated demethylation of histones may be a mechanism for how cells control the establishment of heterochromatin. We find that the abundance of Sir4, a protein required for the assembly of silent chromatin, decreases dramatically during a G1 arrest and therefore tested if changing the levels of Sir4 would also alter the speed of de novo establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. yku70Δ and ubp10Δ cells also speed de novo assembly, and like dot1Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed de novo establishment by liberating Sir4 from telomeres. Deleting RIF1 and RIF2, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced de novo establishment in yku70Δ and ubp10Δ cells, but not in dot1Δ cells, suggesting that YKU70 and UBP10 regulate Sir4 availability by modulating subtelomeric silencing, while DOT1 functions directly to regulate establishment. Our data support a model whereby the demethylation of histone H3 K79 and changes in Sir4 abundance and availability define two rate-limiting steps that regulate de novo assembly of heterochromatin.
Asunto(s)
Silenciador del Gen , Heterocromatina/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Proteínas de Unión al ADN/genética , Epistasis Genética , Fase G1 , Eliminación de Gen , Mutación , Proteínas Nucleares/genética , Proteínas Represoras/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genética , Telómero , Proteínas de Unión a Telómeros/genética , Ubiquitina Tiolesterasa/genéticaRESUMEN
In laboratory yeast strains with Sir2 and Fob1 function, wild-type NAD+ salvage is required for calorie restriction (CR) to extend replicative lifespan. CR does not significantly alter steady state levels of intracellular NAD+ metabolites. However, levels of Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinic acid (NA), are up-regulated during CR. To test whether factors such as NA might be exported by glucose-restricted mother cells to survive later generations, we developed a replicative longevity paradigm in which mother cells are moved after 15 generations on defined media. The experiment reveals that CR mother cells lose the longevity benefit of CR when evacuated from their local environment to fresh CR media. Addition of NA or nicotinamide riboside (NR) allows a moved mother to maintain replicative longevity despite the move. Moreover, conditioned medium from CR-treated cells transmits the longevity benefit of CR to moved mother cells. Evidence suggests the existence of a longevity factor that is dialyzable but is neither NA nor NR, and indicates that Sir2 is not required for the longevity factor to be produced or to act. Data indicate that the benefit of glucose-restriction is transmitted from cell to cell in budding yeast, suggesting that glucose restriction may benefit neighboring cells and not only an individual cell.
Asunto(s)
Saccharomyces cerevisiae/fisiología , Restricción Calórica , Medios de Cultivo Condicionados , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Viabilidad Microbiana , Saccharomyces cerevisiae/citología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiologíaRESUMEN
Saccharomyces cerevisiae provides a well-studied model system for heritable silent chromatin in which a histone-binding protein complex [the SIR (silent information regulator) complex] represses gene transcription in a sequence-independent manner by spreading along nucleosomes, much like heterochromatin in higher eukaryotes. Recent advances in the biochemistry and structural biology of the SIR-chromatin system bring us much closer to a molecular understanding of yeast silent chromatin. Simultaneously, genome-wide approaches have shed light on the biological importance of this form of epigenetic repression. Here, we integrate genetic, structural, and cell biological data into an updated overview of yeast silent chromatin assembly.
Asunto(s)
Cromatina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Acetilación , Cromatina/genética , ADN de Hongos/genética , Silenciador del Gen , Genes Fúngicos , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/fisiología , Modelos Genéticos , Nucleosomas/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Represoras/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Telómero/fisiología , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
Saccharomyces cerevisiae provides a well-studied model system for heritable silent chromatin, in which a nonhistone protein complex--the SIR complex--represses genes by spreading in a sequence-independent manner, much like heterochromatin in higher eukaryotes. The ability to study mutations in histones and to screen genome-wide for mutations that impair silencing has yielded an unparalleled depth of detail about this system. Recent advances in the biochemistry and structural biology of the SIR-chromatin complex bring us much closer to a molecular understanding of how Sir3 selectively recognizes the deacetylated histone H4 tail and demethylated histone H3 core. The existence of appropriate mutants has also shown how components of the silencing machinery affect physiological processes beyond transcriptional repression.
Asunto(s)
Silenciador del Gen , Modelos Genéticos , Saccharomyces cerevisiae/genética , Acetilación , Replicación del ADN/fisiología , Evolución Molecular , Heterocromatina/fisiología , Histonas/metabolismo , Histonas/fisiología , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiologíaRESUMEN
In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Helicasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Telómero/metabolismo , ADN Helicasas/metabolismo , Regulación hacia Abajo , Organismos Modificados Genéticamente , Peptidil Transferasas/metabolismo , Peptidil Transferasas/fisiología , Unión Proteica , Multimerización de Proteína/fisiología , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Sumoilación/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Choque Térmico/fisiología , Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiología , Factores de Transcripción/fisiología , Western Blotting , Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Microscopía Fluorescente , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Factores de Transcripción/metabolismoRESUMEN
In recent years, Hsp90 is found to interact with several telomeric proteins at various phases of cell cycle. The Hsp90 chaperone system controls assembly and disassembly of telomere structures and thus maintains the dynamic state of telomere. Here, for the first time we report that the activity of another telomeric protein Sir2p is modulated by Hsp82, the ortholog of Hsp90 from budding yeast (Saccharomyces cerevisiae). In a temperature sensitive Hsp90 deficient yeast strain (iG170Dhsp82), less abundant Sir2p is observed, resulting in de-repression of telomere silencing and a complete loss of mating type silencing. Intriguingly, over expression of Hsp90, either by exposing cells to heat shock or by introducing HSP82 overexpression plasmid also yields reduced level of Sir2p, with a consequential loss of telomere silencing. Thus, Hsp90 homeostasis maintains the cellular pool of Sir2p and thereby controls the reversible nature of telomere silencing. Interestingly, such regulation is independent of one of its major co-chaperones Sba1 (human ortholog of p23).
Asunto(s)
Silenciador del Gen/fisiología , Proteínas HSP90 de Choque Térmico/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiología , Secuencia de Bases , Western Blotting , Cartilla de ADN , Genes Fúngicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , TelómeroRESUMEN
Discrete regions of the eukaryotic genome assume heritable chromatin structure that is refractory to transcription. In budding yeast, silent chromatin is characterized by the binding of the Silent Information Regulatory (Sir) proteins to unmodified nucleosomes. Using an in vitro reconstitution assay, which allows us to load Sir proteins onto arrays of regularly spaced nucleosomes, we have examined the impact of specific histone modifications on Sir protein binding and linker DNA accessibility. Two typical marks for active chromatin, H3K79(me) and H4K16(ac) decrease the affinity of Sir3 for chromatin, yet only H4K16(ac) affects chromatin structure, as measured by nuclease accessibility. Surprisingly, we found that the Sir2-4 subcomplex, unlike Sir3, has higher affinity for chromatin carrying H4K16(ac). NAD-dependent deacetylation of H4K16(ac) promotes binding of the SIR holocomplex but not of the Sir2-4 heterodimer. This function of H4K16(ac) cannot be substituted by H3K56(ac). We conclude that acetylated H4K16 has a dual role in silencing: it recruits Sir2-4 and repels Sir3. Moreover, the deacetylation of H4K16(ac) by Sir2 actively promotes the high-affinity binding of the SIR holocomplex.
Asunto(s)
Cromatina/metabolismo , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/fisiología , Histonas/metabolismo , Acetilación , Animales , Células Cultivadas , Ensamble y Desensamble de Cromatina/fisiología , Histonas/fisiología , Lisina/metabolismo , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/metabolismo , Sirtuina 2/fisiología , Spodoptera , Levaduras/genética , Levaduras/metabolismoRESUMEN
Protein families are generated by successive rounds of gene duplication and subsequent diversification. However, the paths by which duplicated genes acquire distinct functions are not well characterized. We focused on a pair of duplicated deacetylases from Saccharomyces cerevisiae, Sir2 and Hst1, that subfunctionalized after duplication. As a proxy for the ancestral, nonduplicated deacetylase, we studied Sir2 from another yeast, Kluyveromyces lactis. We compared the interaction domains of these deacetylases for the Sir transcriptional silencing complex, which acts with ScSir2, and the Sum1 repressor, which acts with ScHst1, and found that these interaction domains have been retained over the course of evolution and can be disrupted by simple amino acid substitutions. Therefore, Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.
Asunto(s)
Mutación , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , Evolución Molecular , Kluyveromyces/enzimología , Kluyveromyces/genética , Dominios y Motivos de Interacción de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiologíaRESUMEN
The protein complex known as cohesin binds pericentric regions and other sites of eukaryotic genomes to mediate cohesion of sister chromatids. In budding yeast Saccharomyces cerevisiae, cohesin also binds silent chromatin, a repressive chromatin structure that functionally resembles heterochromatin of higher eukaryotes. We developed a protein-targeting assay to investigate the mechanistic basis for cohesion of silent chromatin domains. Individual silencing factors were tethered to sites where pairing of sister chromatids could be evaluated by fluorescence microscopy. We report that the evolutionarily conserved Sir2 histone deacetylase, an essential silent chromatin component, was both necessary and sufficient for cohesion. The cohesin genes were required, but the Sir2 deacetylase activity and other silencing factors were not. Binding of cohesin to silent chromatin was achieved with a small carboxyl terminal fragment of Sir2. Taken together, these data define a unique role for Sir2 in cohesion of silent chromatin that is distinct from the enzyme's role as a histone deacetylase.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiología , Cromatina/genética , Microscopía Fluorescente , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , CohesinasRESUMEN
Very little is currently known about how nucleotide excision repair (NER) functions at the ends of chromosomes. To examine this, we introduced the URA3 gene into either transcriptionally active or repressed subtelomeric regions of the yeast genome. This enabled us to examine the repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in identical sequences under both circumstances. We found that NER is significantly more efficient in the non-repressed subtelomere than the repressed one. At the non-repressed subtelomere, UV radiation stimulates both histones H3 and H4 acetylation in a similar fashion to that seen at other regions of the yeast genome. These modifications occur regardless of the presence of the Sir2 histone deacetylase. On the other hand, at the repressed subtelomere, where repair is much less efficient, UV radiation is unable to stimulate histone H4 or H3 acetylation in the presence of Sir2. In the absence of Sir2 both of these UV-induced modifications are detected, resulting in a significant increase in NER efficiency in the region. Our experiments reveal that there are instances in the yeast genome where the maintenance of the existing chromatin structures dominates over the action of chromatin modifications associated with efficient NER.
Asunto(s)
Cromatina/química , Reparación del ADN , Silenciador del Gen , Histonas/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiología , Acetilación , Ciclo Celular/efectos de la radiación , Cromatina/metabolismo , Cromosomas Fúngicos , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Nucleasa Microcócica , Dímeros de Pirimidina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de la radiación , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , Transcripción Genética , Rayos UltravioletaRESUMEN
The replicative life span (RLS) of Saccharomyces cerevisiae has been established as a model for the genetic regulation of longevity despite the inherent difficulty of the RLS assay, which requires separation of mother and daughter cells by micromanipulation after every division. Here we present the mother enrichment program (MEP), an inducible genetic system in which mother cells maintain a normal RLS--a median of 36 generations in the diploid MEP strain--while the proliferative potential of daughter cells is eliminated. Thus, the viability of a population over time becomes a function of RLS, and it displays features of a survival curve such as changes in hazard rate with age. We show that viability of mother cells in liquid culture is regulated by SIR2 and FOB1, two opposing regulators of RLS in yeast. We demonstrate that viability curves of these short- and long-lived strains can be easily distinguished from wild type, using a colony formation assay. This provides a simplified screening method for identifying genetic or environmental factors that regulate RLS. Additionally, the MEP can provide a cohort of cells at any stage of their life span for the analysis of age-associated phenotypes. These capabilities effectively remove the hurdles presented by RLS analysis that have hindered S. cerevisiae aging studies since their inception 50 years ago.
Asunto(s)
División Celular/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , División Celular/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Modelos Biológicos , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/genética , Sirtuina 2/fisiología , Factores de TiempoRESUMEN
Heterochromatin, or condensed chromatin, has the potential to encroach into what ordinarily would be euchromatin and repress resident genes. We explore how heterochromatin is restricted to the appropriate regions of the genome, using Saccharomyces cerevisiae as a case study and emphasizing two under-appreciated aspects of silenced chromatin. First, the capacity of silenced chromatin to propagate along a chromosome is limited by the intrinsic instability of the structure. We argue that this limited potential to spread is an important factor restricting silenced chromatin to the vicinity of recruitment sites (silencers). Second, this limited capacity to spread creates the need for additional mechanisms to stabilize silenced chromatin at the required locations. Such mechanisms include the use of multiple silencers and higher-order arrangements of the chromatin fiber. Therefore, to understand how silenced chromatin is restricted to the appropriate genomic locations, researchers must take into account the mechanisms by which silenced chromatin is stabilized in appropriate locations. J. Cell. Physiol. 219: 525-528, 2009. (c) 2009 Wiley-Liss, Inc.
Asunto(s)
Heterocromatina/genética , Heterocromatina/fisiología , Animales , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/fisiología , Silenciador del Gen , Genes Fúngicos , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiologíaAsunto(s)
Reparación del ADN/fisiología , Sirtuinas/fisiología , Acetilación , Envejecimiento/fisiología , Animales , Caenorhabditis elegans/fisiología , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN/fisiología , Proteína Quinasa Activada por ADN/metabolismo , Histonas/metabolismo , Humanos , Ratones , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiologíaAsunto(s)
Replicación del ADN/genética , Amplificación de Genes/genética , Genes de ARNr/genética , ARN no Traducido/genética , Recombinación Genética/genética , Transcripción Genética/genética , Adenosina Trifosfatasas/fisiología , Animales , Proteínas de Unión al ADN/fisiología , Humanos , Complejos Multiproteicos/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Sirtuina 2/fisiologíaRESUMEN
The perinuclear localization of Saccharomyces cerevisiae telomeres provides a useful model for studying mechanisms that control chromosome positioning. Telomeres tend to be localized at the nuclear periphery during early interphase, but following S phase they delocalize and remain randomly positioned within the nucleus. We investigated whether DNA replication causes telomere delocalization from the nuclear periphery. Using live-cell fluorescence microscopy, we show that delaying DNA replication causes a corresponding delay in the dislodgment of telomeres from the nuclear envelope, demonstrating that replication of individual telomeres causes their delocalization. Telomere delocalization is not simply the result of recruitment to a replication factory in the nuclear interior, since we found that telomeric DNA replication can occur either at the nuclear periphery or in the nuclear interior. The telomere-binding complex Ku is one of the factors that localizes telomeres to the nuclear envelope. Using a gene locus tethering assay, we show that Ku-mediated peripheral positioning is switched off after DNA replication. Based on these findings, we propose that DNA replication causes telomere delocalization by triggering stable repression of the Ku-mediated anchoring pathway. In addition to maintaining genetic information, DNA replication may therefore regulate subnuclear organization of chromatin.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/fisiología , Membrana Nuclear/ultraestructura , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/fisiología , Telómero/fisiología , Núcleo Celular , Ciclinas/metabolismo , Fase S , Saccharomyces cerevisiaeRESUMEN
In nearly every organism studied, reduced caloric intake extends life span. In yeast, span extension from dietary restriction is thought to be mediated by the highly conserved, nutrient-responsive target of rapamycin (TOR), protein kinase A (PKA), and Sch9 kinases. These kinases coordinately regulate various cellular processes including stress responses, protein turnover, cell growth, and ribosome biogenesis. Here we show that a specific reduction of 60S ribosomal subunit levels slows aging in yeast. Deletion of genes encoding 60S subunit proteins or processing factors or treatment with a small molecule, which all inhibit 60S subunit biogenesis, are each sufficient to significantly increase replicative life span. One mechanism by which reduced 60S subunit levels leads to life span extension is through induction of Gcn4, a nutrient-responsive transcription factor. Genetic epistasis analyses suggest that dietary restriction, reduced 60S subunit abundance, and Gcn4 activation extend yeast life span by similar mechanisms.