RESUMEN
Recent studies show that antiviral systems are remarkably conserved from bacteria to mammals, demonstrating that unique insights into these systems can be gained by studying microbial organisms. Unlike in bacteria, however, where phage infection can be lethal, no cytotoxic viral consequence is known in the budding yeast Saccharomyces cerevisiae even though it is chronically infected with a double-stranded RNA mycovirus called L-A. This remains the case despite the previous identification of conserved antiviral systems that limit L-A replication. Here, we show that these systems collaborate to prevent rampant L-A replication, which causes lethality in cells grown at high temperature. Exploiting this discovery, we use an overexpression screen to identify antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans. Using a complementary loss of function approach, we identify new antiviral functions for the conserved RNA exonucleases REX2 and MYG1; the SAGA and PAF1 chromatin regulatory complexes; and HSF1, the master transcriptional regulator of the proteostatic stress response. Through investigation of these antiviral systems, we show that L-A pathogenesis is associated with an activated proteostatic stress response and the accumulation of cytotoxic protein aggregates. These findings identify proteotoxic stress as an underlying cause of L-A pathogenesis and further advance yeast as a powerful model system for the discovery and characterization of conserved antiviral systems.
Asunto(s)
Virus Fúngicos , Proteínas de Saccharomyces cerevisiae , Humanos , Animales , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Antivirales , Virus Fúngicos/genética , Virus Fúngicos/metabolismo , ARN Bicatenario , Inmunidad Innata , Mamíferos/genética , Factores de Transcripción/genética , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The Nrd1-Nab3-Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.
Asunto(s)
Histona Desacetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Lisina/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Motivo de Reconocimiento de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Metilación , Unión Proteica , Relación Estructura-ActividadRESUMEN
The programmed release of apoptogenic proteins from mitochondria is a core event of apoptosis, although ancestral roles of this phenomenon are not known. In mammals, one such apoptogenic protein is Endonuclease G (EndoG), a conserved mitochondrial nuclease that fragments the DNA of dying cells. In this work, we show that budding yeast executes meiotically programmed mitochondrial release of an EndoG homolog, Nuc1, during sporulation. In contrast to EndoG's ostensible pro-death function during apoptosis, Nuc1 mitochondrial release is pro-survival, attenuating the cytosolic L-A and Killer double-stranded RNA mycoviruses and protecting meiotic progeny from the catastrophic consequences of their derepression. The protective viral attenuation role of this pathway illuminates a primordial role for mitochondrial release of EndoG, and perhaps of apoptosis itself.
Asunto(s)
Apoptosis/genética , Endonucleasas/genética , Exonucleasas/genética , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Animales , Endodesoxirribonucleasas/genética , Mamíferos , Mitocondrias/enzimología , Mitocondrias/genética , Saccharomycetales/crecimiento & desarrollo , Saccharomycetales/virología , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
Viruses and other genetic parasites are present in virtually all forms of life. This chronic condition has led to diverse host cell adaptations such as CRISPR and RNAi, whose functions attenuate these parasites. It is hypothesized that programmed cell death (PCD) is an additional adaptation whose origins reside in viral defense. A core event of apoptotic PCD is the regulated release of mitochondrial inter-membrane space proteins into the cytosol, following which these apoptogenic proteins bring about the demise of the cell. The most well studied example of this is found in animals, where the release of mitochondrial cytochrome C nucleates the formation of the apoptosome, which then activates caspase mediated cell death. The release of mitochondrial proteins contributes to PCD in diverse organisms lacking the apoptosome, indicating that regulated mitochondrial release predates the evolution of canonical apoptosis. Using the budding yeast Saccharomyces cerevisiae, we recently confirmed an early study showing that Nuc1, a homolog of the mitochondrial apoptotic driver protein Endonuclease G, attenuates cytosolic double stranded RNA (dsRNA) viruses, which are endemic to yeast and many other organisms. Viral attenuation by Nuc1 occurs most prominently during meiosis and in association with its developmentally programmed relocation from the mitochondria to the cytosol. Intriguingly, meiotic viral attenuation by Nuc1 occurs within the context of meiotic PCD of the superfluous mother cell that we have also discovered. These findings are discussed here.
RESUMEN
Transient exposures to environmental stresses induce altered physiological states in exposed cells that persist after the stresses have been removed. These states, referred to as cellular memory, can even be passed on to daughter cells and may thus be thought of as embodying a form of epigenetic inheritance. We find that meiotically produced spores in the budding yeast S. cerevisiae possess a state of heightened stress resistance that, following their germination, persists for numerous mitotic generations. As yeast meiotic development is essentially a starvation response that a/alpha diploid cells engage, we sought to model this phenomenon by subjecting haploid cells to starvation conditions. We find also that haploid cells exposed to glucose withdrawal acquire a state of elevated stress resistance that persists after the reintroduction of these cells to glucose-replete media. Following release from lengthy durations of glucose starvation, we confirm that this physiological state of enhanced stress resistance is propagated in descendants of the exposed cells through two mitotic divisions before fading from the population. In both haploid starved cells and diploid produced meiotic spores we show that their cellular memories are not attributable to trehalose, a widely regarded stress protectant that accumulates in these cell types. Moreover, the transiently heritable stress resistant state induced by glucose starvation in haploid cells is independent of the Msn2/4 transcription factors, which are known to program cellular memory induced by exposure of cells to NaCl. Our findings identify new developmentally and nutritionally induced states of cellular memory that exhibit striking degrees of persistence and mitotic heritability.
RESUMEN
Much of euchromatin regulation occurs through reversible methylation of histone H3 lysine-4 and lysine-36 (H3K4me and H3K36me). Using the budding yeast Saccharomyces cerevisiae, we previously found that levels of H3K4me modulated temperature sensitive alleles of the transcriptional elongation complex Spt6-Spn1 through an unknown H3K4me effector pathway. Here we identify the Rpd3S histone deacetylase complex as the H3K4me effector underlying these Spt6-Spn1 genetic interactions. Exploiting these Spt6-Spn1 genetic interactions, we show that H3K4me and H3K36me collaboratively impact Rpd3S function in an opposing manner. H3K36me is deposited by the histone methyltransferase Set2 and is known to promote Rpd3S function at RNA PolII transcribed open reading frames. Using genetic epistasis experiments, we find that mutations perturbing the Set2-H3K36me-Rpd3S pathway suppress the growth defects caused by temperature sensitive alleles of SPT6 and SPN1, illuminating that this pathway antagonizes Spt6-Spn1 Using these sensitive genetic assays, we also identify a role for H3K4me in antagonizing Rpd3S that functions through the Rpd3S subunit Rco1, which is known to bind H3 N-terminal tails in a manner that is prevented by H3K4me. Further genetic experiments reveal that the H3K4 and H3K36 demethylases JHD2 and RPH1 mediate this combinatorial control of Rpd3S. Finally, our studies also show that the Rpd3L complex, which acts at promoter-proximal regions of PolII transcribed genes, counters Rpd3S for genetic modulation of Spt6-Spn1, and that these two Rpd3 complexes balance the activities of each other. Our findings present the first evidence that H3K4me and H3K36me act combinatorially to control Rpd3S.
Asunto(s)
Chaperonas de Histonas/genética , Histona Desacetilasas/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Elongación Transcripcional/genética , Histonas/metabolismo , MetilaciónRESUMEN
Set1 and Jhd2 regulate the methylation state of histone H3 lysine-4 (H3K4me) through their opposing methyltransferase and demethylase activities in the budding yeast Saccharomyces cerevisiae H3K4me associates with actively transcribed genes and, like both SET1 and JHD2 themselves, is known to regulate gene expression diversely. It remains unclear, however, if Set1 and Jhd2 act solely through H3K4me. Relevantly, Set1 methylates lysine residues in the kinetochore protein Dam1 while genetic studies of the S. pombe SET1 ortholog suggest the existence of non-H3K4 Set1 targets relevant to gene regulation. We interrogated genetic interactions of JHD2 and SET1 with essential genes involved in varied aspects of the transcription cycle. Our findings implicate JHD2 in genetic inhibition of the histone chaperone complexes Spt16-Pob3 (FACT) and Spt6-Spn1 This targeted screen also revealed that JHD2 inhibits the Nrd1-Nab3-Sen1 (NNS) transcription termination complex. We find that while Jhd2's impact on these transcription regulatory complexes likely acts via H3K4me, Set1 governs the roles of FACT and NNS through opposing H3K4-dependent and -independent functions. We also identify diametrically opposing consequences for mutation of H3K4 to alanine or arginine, illuminating that caution must be taken in interpreting histone mutation studies. Unlike FACT and NNS, detailed genetic studies suggest an H3K4me-centric mode of Spt6-Spn1 regulation by JHD2 and SET1 Chromatin immunoprecipitation and transcript quantification experiments show that Jhd2 opposes the positioning of a Spt6-deposited nucleosome near the transcription start site of SER3, a Spt6-Spn1 regulated gene, leading to hyper-induction of SER3 In addition to confirming and extending an emerging role for Jhd2 in the control of nucleosome occupancy near transcription start sites, our findings suggest some of the chromatin regulatory functions of Set1 are independent of H3K4 methylation.
Asunto(s)
Cromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Alelos , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Metilación , Modelos Genéticos , Nucleosomas/metabolismo , Subunidades de Proteína/metabolismo , Saccharomycetales/genética , Supresión Genética , Temperatura , Sitio de Iniciación de la TranscripciónRESUMEN
Histone demethylation by Jumonji-family proteins is coupled with the decarboxylation of α-ketoglutarate (αKG) to yield succinate, prompting hypotheses that their activities are responsive to levels of these metabolites in the cell. Consistent with this paradigm we show here that the Saccharomyces cerevisiae Jumonji demethylase Jhd2 opposes the accumulation of H3K4me3 in fermenting cells only when they are nutritionally manipulated to contain an elevated αKG/succinate ratio. We also find that Jhd2 opposes H3K4me3 in respiratory cells that do not exhibit such an elevated αKG/succinate ratio. While jhd2∆ caused only limited gene expression defects in fermenting cells, transcript profiling and physiological measurements show that JHD2 restricts mitochondrial respiratory capacity in cells grown in non-fermentable carbon in an H3K4me-dependent manner. In association with these phenotypes, we find that JHD2 limits yeast proliferative capacity under physiologically challenging conditions as measured by both replicative lifespan and colony growth on non-fermentable carbon. JHD2's impact on nutrient response may reflect an ancestral role of its gene family in mediating mitochondrial regulation.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Mitocondrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Replicación del ADN , Desmetilación , Histonas/genética , Histona Demetilasas con Dominio de Jumonji/genética , Ácidos Cetoglutáricos/metabolismo , Lisina/genética , Mitocondrias/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Ácido Succínico/metabolismo , Transcripción GenéticaRESUMEN
In this issue of Developmental Cell, Tan et al. (2015) describe an elegant mechanism functioning during sporulation in Bacillus subtilis. Their findings suggest that quality control purges unfit spores via programmed cell death, providing further insight into the utility of this phenomenon in unicellular organisms.
Asunto(s)
Apoptosis , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Comunicación Celular , Esporas Bacterianas/fisiologíaRESUMEN
The gametogenesis program of the budding yeast Saccharomyces cerevisiae, also known as sporulation, employs unusual internal meiotic divisions, after which all four meiotic products differentiate within the parental cell. We showed previously that sporulation is typically accompanied by the destruction of discarded immature meiotic products through their exposure to proteases released from the mother cell vacuole, which undergoes an apparent programmed rupture. Here we demonstrate that vacuolar rupture contributes to de facto programmed cell death (PCD) of the meiotic mother cell itself. Meiotic mother cell PCD is accompanied by an accumulation of depolarized mitochondria, organelle swelling, altered plasma membrane characteristics, and cytoplasmic clearance. To ensure that the gametes survive the destructive consequences of developing within a cell that is executing PCD, we hypothesized that PCD is restrained from occurring until spores have attained a threshold degree of differentiation. Consistent with this hypothesis, gene deletions that perturb all but the most terminal postmeiotic spore developmental stages are associated with altered PCD. In these mutants, meiotic mother cells exhibit a delay in vacuolar rupture and then appear to undergo an alternative form of PCD associated with catastrophic consequences for the underdeveloped spores. Our findings reveal yeast sporulation as a context of bona fide PCD that is developmentally coordinated with gamete differentiation.
Asunto(s)
Apoptosis , Meiosis , Saccharomyces cerevisiae/fisiología , Esporas Fúngicas/fisiología , Eliminación de Gen , Potencial de la Membrana Mitocondrial , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Esporas Fúngicas/citología , Esporas Fúngicas/genéticaRESUMEN
Studies of the budding yeast Saccharomyces cerevisiae have provided many of the most important insights into the mechanisms of autophagy, which are common to all eukaryotes. However, investigation of yeast self-destruction pathways, including autophagy and programmed cell death, has been almost exclusively restricted to cells undergoing vegetative growth, leaving very little exploration of their functions during developmental transitions in the yeast life cycle. We have recently discovered that whole nuclei are subject to programmed destruction during yeast gametogenesis. Programmed nuclear destruction (PND) possesses characteristics of apoptosis in the form of DNA cleavage by endonuclease G, and involves bulk protein turnover through an unusual autophagic pathway involving lysis of the vacuole rather than delivery of components to it through macroautophagy. We thus illuminate an example of developmentally programmed cellular "self-eating" in yeast, which is associated with the rupture of a lytic organelle, reminiscent of programmed cell death mechanisms in plants and animals.
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Autofagia , Núcleo Celular/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Animales , Núcleo Celular/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/citología , Esporas Fúngicas/ultraestructuraRESUMEN
Gametes are among the most highly specialized cells produced during development. Although gametogenesis culminates in transcriptional quiescence in plants and animals, regulatory mechanisms controlling this are unknown. Here, we confirm that gamete differentiation in the single-celled yeast Saccharomyces cerevisiae is accompanied by global transcriptional shutoff following the completion of meiosis. We show that Jhd2, a highly conserved JARID1-family histone H3K4 demethylase, activates protein-coding gene transcription in opposition to this programmed transcriptional shutoff, sustaining the period of productive transcription during spore differentiation. Moreover, using genome-wide nucleosome, H3K4me, and transcript mapping experiments, we demonstrate that JHD2 globally represses intergenic noncoding transcription during this period. The widespread transcriptional defects of JHD2 mutants are associated with precocious differentiation and the production of stress-sensitive spores, demonstrating that Jhd2 regulation of the global postmeiotic transcriptional program is critical for the production of healthy meiotic progeny.
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Gametogénesis/genética , Gametogénesis/fisiología , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Epigénesis Genética , Genes Fúngicos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Meiosis , Metilación , Mutación , Nucleosomas/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejo Shelterina , Esporas Fúngicas/genética , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Autophagy controls cellular catabolism in diverse eukaryotes and modulates programmed cell death in plants and animals. While studies of the unicellular yeast Saccharomyces cerevisiae have provided fundamental insights into the mechanisms of autophagy, the roles of cell death pathways in yeast are less well understood. Here, we describe widespread developmentally programmed nuclear destruction (PND) events that occur during yeast gametogenesis. PND is executed through apoptotic-like DNA fragmentation in coordination with an unusual form of autophagy that is most similar to mammalian lysosomal membrane permeabilization and mega-autophagy, a form of plant autophagic cell death. Undomesticated strains execute gametogenic PND broadly in maturing colonies to the apparent benefit of sibling cells, confirming its prominence during the yeast life cycle. Our results reveal that diverse cell-death-related processes converge during gametogenesis in a microbe distantly related to plants or animals, highlighting gametogenesis as a process during which programmed cell death mechanisms may have evolved.
Asunto(s)
Núcleo Celular/fisiología , Fragmentación del ADN , Gametogénesis/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/fisiología , Esporas Fúngicas/fisiología , Animales , Apoptosis/fisiología , Autofagia/fisiología , Núcleo Celular/ultraestructura , Gametogénesis en la Planta/fisiología , Lisosomas/fisiología , Mamíferos , Saccharomyces cerevisiae/citología , Esporas Fúngicas/ultraestructuraRESUMEN
In Saccharomyces cerevisiae, several nonessential mechanisms including histone variant H2A.Z deposition and transcription-associated histone H3 methylation antagonize the local spread of Sir-dependent silent chromatin into adjacent euchromatic regions. However, it is unclear how and where these factors cooperate. To probe this question, we performed systematic genetic array screens for gene deletions that cause a synthetic growth defect in an htz1Delta mutant but not in an htz1Delta sir3Delta double mutant. Of the four genes identified, three, SET1, SWD1, and SWD3, encode components of the Set1 complex, which catalyzes the methylation of histone H3 on lysine 4 (H3-K4), a highly conserved modification that occurs in the coding sequences of transcribed genes. Using microarray-based transcriptional profiling, we find that H2A.Z and Set1 cooperate to prevent Sir-dependent repression of a large number of genes located across the genome, rather than the local effects reported previously for the individual mechanisms. This global, redundant function appears to be direct: using a DamID chromatin profiling method, we demonstrate ectopic association of Sir3 and Sir4 in htz1Delta set1Delta mutants at loci distant from silent chromatin domains. Antisilencing mechanisms may therefore cooperate to play a considerably broader role in regulating genome-wide transcription than previously thought.
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Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismo , Eucromatina/metabolismo , Genoma Fúngico/genética , N-Metiltransferasa de Histona-Lisina , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Regulación hacia ArribaRESUMEN
In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.
Asunto(s)
Eucromatina/genética , Genes Fúngicos , Variación Genética , Histonas/genética , Acetilación , Sustitución de Aminoácidos , Arginina/metabolismo , Sitios de Unión , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Cromosomas , Codón Iniciador , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Intergénico/genética , ADN Intergénico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Heterocromatina/metabolismo , Histonas/metabolismo , Análisis por Micromatrices , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The conserved histone variant H2A.Z functions in euchromatin to antagonize the spread of heterochromatin. The mechanism by which histone H2A is replaced by H2A.Z in the nucleosome is unknown. We identified a complex containing 13 different polypeptides associated with a soluble pool of H2A.Z in Saccharomyces cerevisiae. This complex was designated SWR1-Com in reference to the Swr1p subunit, a Swi2/Snf2-paralog. Swr1p and six other subunits were found only in SWR1-Com, whereas six other subunits were also found in the NuA4 histone acetyltransferase and/or the Ino80 chromatin remodeling complex. H2A.Z and SWR1 were essential for viability of cells lacking the EAF1 component of NuA4, pointing to a close functional connection between these two complexes. Strikingly, chromatin immunoprecipitation analysis of cells lacking Swr1p, the presumed ATPase of the complex, revealed a profound defect in the deposition of H2A.Z at euchromatic regions that flank the silent mating type cassette HMR and at 12 other chromosomal sites tested. Consistent with a specialized role for Swr1p in H2A.Z deposition, the majority of the genome-wide transcriptional defects seen in swr1Delta cells were also found in htz1Delta cells. These studies revealed a novel role for a member of the ATP-dependent chromatin remodeling enzyme family in determining the region-specific histone subunit composition of chromatin in vivo and controlling the epigenetic state of chromatin. Metazoan orthologs of Swr1p (Drosophila Domino; human SRCAP and p400) may have analogous functions.
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Adenosina Trifosfatasas/fisiología , Eucromatina/química , Regulación Fúngica de la Expresión Génica , Histonas/química , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/fisiología , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Supervivencia Celular , Cromatina/química , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Drosophila , Epigénesis Genética , Proteínas Fúngicas/química , Genoma Fúngico , Heterocromatina/química , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Boundary elements hinder the spread of heterochromatin, yet these sites do not fully account for the preservation of adjacent euchromatin. Histone variant H2A.Z (Htz1 in yeast) replaces conventional H2A in many nucleosomes. Microarray analysis revealed that HTZ1-activated genes cluster near telomeres. The reduced expression of most of these genes in htz1Delta cells was reversed by the deletion of SIR2 (sir2Delta) suggesting that H2A.Z antagonizes telomeric silencing. Other Htz1-activated genes flank the silent HMR mating-type locus. Their requirement for Htz1 can be bypassed by sir2Delta or by a deletion encompassing the silencing nucleation sites in HMR. In htz1Delta cells, Sir2 and Sir3 spread into flanking euchromatic regions, producing changes in histone H4 acetylation and H3 4-methylation indicative of ectopic heterochromatin formation. Htz1 is enriched in these euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin. Thus, euchromatin and heterochromatin each contains components that antagonize switching to the opposite chromatin state.