RESUMEN
Transcriptional silencing in Saccharomyces cerevisiae involves the generation of a chromatin state that stably represses transcription. Using multiple reporter assays, a diverse set of upstream activating sequence enhancers and core promoters were investigated for their susceptibility to silencing. We show that heterochromatin stably silences only weak and stress-induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements, and the partial repression of these elements did not result in bistable expression states. Permutation analysis of enhancers and promoters indicates that both elements are targets of repression. Chromatin remodelers help specific regulatory elements to resist repression, most probably by altering nucleosome mobility and changing transcription burst duration. The strong enhancers/promoters can be repressed if silencer-bound Sir1 is increased. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating-type gene regulatory elements but not strong housekeeping gene regulatory sequences.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Heterocromatina , Regiones Promotoras Genéticas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Heterocromatina/metabolismo , Heterocromatina/genética , Regiones Promotoras Genéticas/genética , Elementos de Facilitación Genéticos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
The interplay between nucleosomes and transcription factors leads to programs of gene expression. Transcriptional silencing involves the generation of a chromatin state that represses transcription and is faithfully propagated through DNA replication and cell division. Using multiple reporter assays, including directly visualizing transcription in single cells, we investigated a diverse set of UAS enhancers and core promoters for their susceptibility to heterochromatic gene silencing. These results show that heterochromatin only stably silences weak and stress induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements and the partial repression did not result in bistable expression states. Permutation analysis of different UAS enhancers and core promoters indicate that both elements function together to determine the susceptibility of regulatory sequences to repression. Specific histone modifiers and chromatin remodellers function in an enhancer specific manner to aid these elements to resist repression suggesting that Sir proteins likely function in part by reducing nucleosome mobility. We also show that the strong housekeeping regulatory elements can be repressed if silencer bound Sir1 is increased, suggesting that Sir1 is a limiting component in silencing. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating type gene regulatory elements but not strong housekeeping gene regulatory sequences which could help explain why these genes are often found at the boundaries of silenced domains.
RESUMEN
RNA polymerase III (Pol III) includes two alternate isoforms, defined by mutually exclusive incorporation of subunit POLR3G (RPC7α) or POLR3GL (RPC7ß), in mammals. The contributions of POLR3G and POLR3GL to transcription potential has remained poorly defined. Here, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. Particularly sensitive is snaR-A, a small noncoding RNA implicated in cancer proliferation and metastasis. Analysis of Pol III isoform biases and downstream chromatin features identifies loss of POLR3G and snaR-A during differentiation, and conversely, re-establishment of POLR3G gene expression and SNAR-A gene features in cancer contexts. Our results support a model in which Pol III identity functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.
Asunto(s)
Neoplasias , ARN Pequeño no Traducido , Animales , Cromatina , Humanos , Mamíferos/genética , Neoplasias/genética , Isoformas de Proteínas/genética , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismoRESUMEN
Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.
Asunto(s)
Silenciador del Gen/fisiología , Histonas/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Acetilación , Cromatina/metabolismo , Heterocromatina/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/genética , Sirtuina 2/metabolismo , Telómero/metabolismoRESUMEN
The genome is packaged and organized in an ordered, nonrandom manner, and specific chromatin segments contact nuclear substructures to mediate this organization. tRNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the roles of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacked any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long-range HML-HMR heterochromatin clustering with concomitant effects on gene silencing. We propose that the tDNAs primarily affect local chromatin structure, which results in effects on long-range chromosome architecture.
Asunto(s)
Cromatina/metabolismo , Cromatina/ultraestructura , ARN de Transferencia/genética , Sitios de Unión , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Ensamble y Desensamble de Cromatina , Cromosomas/genética , Cromosomas/metabolismo , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción TFIII/metabolismoRESUMEN
Silenced heterochromatin influences all nuclear processes including chromosome structure, nuclear organization, transcription, replication, and repair. Proteins that mediate silencing affect all of these nuclear processes. Similarly proteins involved in replication, repair, and chromosome structure play a role in the formation and maintenance of silenced heterochromatin. In this chapter we describe a handful of simple tools and methods that can be used to study the atypical role of proteins in gene silencing.
Asunto(s)
Proteínas de Ciclo Celular/genética , Silenciador del Gen , Biología Molecular/métodos , Proteínas Nucleares/genética , Proteínas Cromosómicas no Histona , Cromosomas/genética , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Heterocromatina/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Heterochromatin formation and nuclear organization are important in gene regulation and genome fidelity. Proteins involved in gene silencing localize to sites of damage and some DNA repair proteins localize to heterochromatin, but the biological importance of these correlations remains unclear. In this study, we examined the role of double-strand-break repair proteins in gene silencing and nuclear organization. We find that the ATM kinase Tel1 and the proteins Mre11 and Esc2 can silence a reporter gene dependent on the Sir, as well as on other repair proteins. Furthermore, these proteins aid in the localization of silenced domains to specific compartments in the nucleus. We identify two distinct mechanisms for repair protein-mediated silencing-via direct and indirect interactions with Sir proteins, as well as by tethering loci to the nuclear periphery. This study reveals previously unknown interactions between repair proteins and silencing proteins and suggests insights into the mechanism underlying genome integrity.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromosomas Fúngicos/metabolismo , Reparación del ADN , Heterocromatina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Condensin is enriched in the pericentromere of budding yeast chromosomes where it is constrained to the spindle axis in metaphase. Pericentric condensin contributes to chromatin compaction, resistance to microtubule-based spindle forces, and spindle length and variance regulation. Condensin is clustered along the spindle axis in a heterogeneous fashion. We demonstrate that pericentric enrichment of condensin is mediated by interactions with transfer ribonucleic acid (tRNA) genes and their regulatory factors. This recruitment is important for generating axial tension on the pericentromere and coordinating movement between pericentromeres from different chromosomes. The interaction between condensin and tRNA genes in the pericentromere reveals a feature of yeast centromeres that has profound implications for the function and evolution of mitotic segregation mechanisms.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Hidroliasas/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Mitosis/fisiología , Complejos Multiproteicos/metabolismo , ARN de Transferencia/genética , Ribonucleoproteínas Nucleares Pequeñas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/citología , Huso Acromático/metabolismo , Adenosina Trifosfatasas/análisis , Centrosoma/metabolismo , Centrosoma/ultraestructura , Cromatina/ultraestructura , Proteínas de Unión al ADN/análisis , Hidroliasas/análisis , Hidroliasas/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/análisis , Ribonucleoproteínas Nucleares Pequeñas/análisis , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Huso Acromático/ultraestructuraRESUMEN
The eukaryotic genome is highly organized in the nucleus, and this organization affects various nuclear processes. However, the molecular details of higher-order organization of chromatin remain obscure. In the present study, we show that the Saccharomyces cerevisiae silenced loci HML and HMR cluster in three-dimensional space throughout the cell cycle and independently of the telomeres. Long-range HML-HMR interactions require the homologous recombination (HR) repair pathway and phosphorylated H2A (γ-H2A). γ-H2A is constitutively present at silenced loci in unperturbed cells, its localization requires heterochromatin, and it is restricted to the silenced domain by the transfer DNA boundary element. SMC proteins and Scc2 localize to the silenced domain, and Scc2 binding requires the presence of γ-H2A. These findings illustrate a novel pathway for heterochromatin organization and suggest a role for HR repair proteins in genomic organization.
Asunto(s)
Reparación del ADN/genética , Proteínas HSP70 de Choque Térmico/genética , Histonas/genética , Proteínas Mitocondriales/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Telómero/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromosomas Fúngicos/genética , Roturas del ADN , Silenciador del Gen/fisiología , Genes del Tipo Sexual de los Hongos/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Mitocondriales/metabolismo , Fosforilación/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/metabolismoRESUMEN
tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short interspersed nuclear elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer - blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vice versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. This article is part of a Special Issue entitled: Transcription by Odd Pols.
Asunto(s)
Elementos Aisladores , Elementos de Nucleótido Esparcido Corto , Factores de Transcripción TFIII/metabolismo , Animales , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Humanos , ARN de Transferencia/biosíntesis , ARN de Transferencia/genética , Levaduras/genética , Levaduras/metabolismoRESUMEN
Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes.
Asunto(s)
Elementos Aisladores/genética , ARN de Transferencia/genética , Animales , Línea Celular , Cromatina/genética , Cromosomas Humanos Par 17/genética , Biología Computacional/métodos , ADN de Hongos/genética , ADN de Hongos/metabolismo , Elementos de Facilitación Genéticos/genética , Silenciador del Gen , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Mamíferos/genética , Unión Proteica , ARN Polimerasa III/metabolismo , Schizosaccharomyces/genética , Alineación de Secuencia , Sintenía , Factores de Transcripción TFIII/metabolismo , Transcripción Genética/genética , TransgenesRESUMEN
The organization of chromatin domains in the nucleus is an important factor in gene regulation. In eukaryotic nuclei, transcriptionally silenced chromatin clusters at the nuclear periphery while transcriptionally poised chromatin resides in the nuclear interior. Recent studies suggest that nuclear pore proteins (NUPs) recruit loci to nuclear pores to aid in insulation of genes from silencing and during gene activation. We investigated the role of NUPs at a native yeast insulator and show that while NUPs localize to the native tDNA insulator adjacent to the silenced HMR domain, loss of pore proteins does not compromise insulation. Surprisingly we find that NUPs contribute to silencing at HMR and are able to restore silencing to a silencing-defective HMR allele when tethered to the locus. We show that the perinuclear positioning of heterochromatin is important for the NUP-mediated silencing effect and find that loss of NUPs result in decreased localization of HMR to the nuclear periphery. We also show that loss of telomeric tethering pathways does not eliminate NUP localization to HMR, suggesting that NUPs may mediate an independent pathway for HMR association with the nuclear periphery. We propose that localization of NUPs to the tDNA insulator at HMR helps maintain the intranuclear position of the silent locus, which in turn contributes to the fidelity of silencing at HMR.
Asunto(s)
Núcleo Celular/metabolismo , Silenciador del Gen , Sitios Genéticos/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , ADN de Hongos/metabolismo , Heterocromatina/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/metabolismoRESUMEN
Insulators prevent promiscuous gene regulation by restricting the action of enhancers and silencers. Recent studies have revealed a number of similarities between insulators and promoters, including binding of specific transcription factors, chromatin-modification signatures and localization to specific subnuclear positions. We propose that enhancer-blockers and silencing barrier-insulators might have evolved as specialized derivatives of promoters and that the two types of element use related mechanisms to mediate their distinct functions. These insights can help to reconcile different models of insulator action.
Asunto(s)
Evolución Molecular , Regulación de la Expresión Génica/genética , Elementos Aisladores/fisiología , Regiones Promotoras Genéticas/fisiología , Animales , Mapeo Cromosómico , Elementos de Facilitación Genéticos/genética , Elementos de Facilitación Genéticos/fisiología , Humanos , Elementos Aisladores/genética , Modelos Biológicos , Modelos Genéticos , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiologíaRESUMEN
DNA in eukaryotes is invariably present as a complex with histone and non-histone proteins called chromatin. These proteins play an important role in the proper regulation of genes during development and differentiation. Transcription factors and the covalent modifications of DNA, histone and non-histone proteins establish an epigenetic state that is heritable and which does not involve a change in genotype. The heritability of transcription states through cell division brings up specific questions: How are epigenetic marks established and re-established in the daughter cells following DNA replication and mitosis? In this article we explore what is known of the cell cycle dependence of epigenetic inheritance with particular emphasis on yeast loci and discuss the role of specific proteins responsible for the establishment and maintenance of these states.
Asunto(s)
Patrón de Herencia/genética , Elementos Aisladores/genética , ARN de Transferencia/genética , Transcripción Genética/genética , Replicación del ADN/genética , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
Chromatin insulators separate active from repressed chromatin domains. In yeast the RNA pol III transcription machinery bound to tRNA genes function with histone acetylases and chromatin remodelers to restrict the spread of heterochromatin. Our results collectively demonstrate that binding of TFIIIC is necessary for insulation but binding of TFIIIB along with TFIIIC likely improves the probability of complex formation at an insulator. Insulation by this transcription factor occurs in the absence of RNA polymerase III or polymerase II but requires specific histone acetylases and chromatin remodelers. This analysis identifies a minimal set of factors required for insulation.
Asunto(s)
Elementos Aisladores/fisiología , ARN de Transferencia/genética , Factores de Transcripción TFIII/fisiología , Transcripción Genética/genética , Ensamble y Desensamble de Cromatina/genética , Mapeo Cromosómico , Histona Acetiltransferasas/metabolismo , Organismos Modificados Genéticamente , Multimerización de Proteína , ARN de Transferencia/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Factores de Transcripción TFIII/metabolismo , Transcripción Genética/fisiología , Levaduras/genéticaRESUMEN
Insulators bind transcription factors and use chromatin remodellers and modifiers to mediate insulation. In this report, we identified proteins required for the efficient formation and maintenance of a specialized chromatin structure at the yeast tRNA insulator. The histone acetylases, SAS-I and NuA4, functioned in insulation, independently of tRNA and did not participate in the formation of the hypersensitive site at the tRNA. In contrast, DNA polymerase epsilon, functioned with the chromatin remodeller, Rsc, and the histone acetylase, Rtt109, to generate a histone-depleted region at the tRNA insulator. Rsc and Rtt109 were required for efficient binding of TFIIIB to the tRNA insulator, and the bound transcription factor and Rtt109 in turn were required for the binding of Rsc to tRNA. Robust insulation during growth and cell division involves the formation of a hypersensitive site at the insulator during chromatin maturation together with competition between acetylases and deacetylases.
Asunto(s)
Cromatina/química , ADN Polimerasa II/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Elementos Aisladores , ARN de Transferencia/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Histona Acetiltransferasas/genética , Histonas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Heterochromatin resides near yeast telomeres and at the cryptic mating-type loci, HML and HMR, where it silences transcription of the alpha- and a-mating-type genes, respectively. Ku is a conserved DNA end-binding protein that binds telomeres and regulates silencing in yeast. The role of Ku in silencing is thought to be limited to telomeric silencing. Here, we tested whether Ku contributes to silencing at HML or HMR. Mutant analysis revealed that yKu70 and Sir1 act collectively to silence the mating-type genes at HML and HMR. In addition, loss of yKu70 function leads to expression of different reporter genes inserted at HMR. Quantitative chromatin-immunoprecipitation experiments revealed that yKu70 binds to HML and HMR and that binding of Ku to these internal loci is dependent on Sir4. The interaction between yKu70 and Sir4 was characterized further and found to be dependent on Sir2 but not on Sir1, Sir3, or yKu80. These observations reveal that, in addition to its ability to bind telomeric DNA ends and aid in the silencing of genes at telomeres, Ku binds to internal silent loci via protein-protein interactions and contributes to the efficient silencing of these loci.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Genes del Tipo Sexual de los Hongos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Inmunoprecipitación de Cromatina , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Reacción en Cadena de la Polimerasa , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Telómero/metabolismo , Transcripción GenéticaRESUMEN
The packaging of eukaryotic DNA into chromatin is likely to be crucial for the maintenance of genomic integrity. Histone acetylation and deacetylation, which alter chromatin accessibility, have been implicated in DNA damage tolerance. Here we show that Schizosaccharomyces pombe Hst4, a homolog of histone deacetylase Sir2, participates in S-phase-specific DNA damage tolerance. Hst4 was essential for the survival of cells exposed to the genotoxic agent methyl methanesulfonate (MMS) as well as for cells lacking components of the DNA damage checkpoint pathway. It was required for the deacetylation of histone H3 core domain residue lysine 56, since a strain with a point mutation of its catalytic domain was unable to deacetylate this residue in vivo. Hst4 regulated the acetylation of H3 K56 and was itself cell cycle regulated. We also show that MMS treatment resulted in increased acetylation of histone H3 lysine 56 in wild-type cells and hst4Delta mutants had constitutively elevated levels of histone H3 K56 acetylation. Interestingly, the level of expression of Hst4 decreased upon MMS treatment, suggesting that the cell regulates access to the site of DNA damage by changing the level of this protein. Furthermore, we find that the phenotypes of both K56Q and K56R mutants of histone H3 were similar to those of hst4Delta mutants, suggesting that proper regulation of histone acetylation is important for DNA integrity. We propose that Hst4 is a deacetylase involved in the restoration of chromatin structure following the S phase of cell cycle and DNA damage response.
Asunto(s)
Daño del ADN , Regulación Fúngica de la Expresión Génica , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Acetilación , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Codón sin Sentido , Enzimas Reparadoras del ADN/metabolismo , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/genética , Histonas/antagonistas & inhibidores , Histonas/genética , Mutágenos/farmacología , Fenotipo , Procesamiento Proteico-Postraduccional , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/antagonistas & inhibidores , Proteínas de Schizosaccharomyces pombe/genética , Rayos UltravioletaRESUMEN
Gene regulation involves long-range communication between silencers, enhancers, and promoters. In Saccharomyces cerevisiae, silencers flank transcriptionally repressed genes to mediate regional silencing. Silencers recruit the Sir proteins, which then spread along chromatin to encompass the entire silenced domain. In this report we have employed a boundary trap assay, an enhancer activity assay, chromatin immunoprecipitations, and chromosome conformation capture analyses to demonstrate that the two HMR silencer elements are in close proximity and functionally communicate with one another in vivo. We further show that silencing is necessary for these long-range interactions, and we present models for Sir-mediated silencing based upon these results.