RESUMO
SWI/SNF-family chromatin remodeling complexes, such as S. cerevisiae RSC, slide and eject nucleosomes to regulate transcription. Within nucleosomes, stiff DNA sequences confer spontaneous partial unwrapping, prompting whether and how SWI/SNF-family remodelers are specialized to remodel partially-unwrapped nucleosomes. RSC1 and RSC2 are orthologs of mammalian PBRM1 (polybromo) which define two separate RSC sub-complexes. Remarkably, in vitro the Rsc1-containing complex remodels partially-unwrapped nucleosomes much better than does the Rsc2-containing complex. Moreover, a rsc1Δ mutation, but not rsc2Δ, is lethal with histone mutations that confer partial unwrapping. Rsc1/2 isoforms both cooperate with the DNA-binding proteins Rsc3/30 and the HMG protein, Hmo1, to remodel partially-unwrapped nucleosomes, but show differential reliance on these factors. Notably, genetic impairment of these factors strongly reduces the expression of genes with wide nucleosome-deficient regions (e.g., ribosomal protein genes), known to harbor partially-unwrapped nucleosomes. Taken together, Rsc1/2 isoforms are specialized through composition and interactions to manage and remodel partially-unwrapped nucleosomes.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Nucleossomos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
ISWI family chromatin remodelers typically organize nucleosome arrays, while SWI/SNF family remodelers (RSC) typically disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex or mutations in the 'basic patch' of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism and reveal different mechanisms at two different promoter architectures.
Assuntos
Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/metabolismo , Ligação Competitiva , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Histonas/genética , Histonas/metabolismo , Família Multigênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Trimethylation of lysine 4 of histone H3 occurs at the 5' end of active genes and is catalyzed by Set1 in Saccharomyces cerevisiae. Trimethylation requires histone H2B ubiquitylation and the PAF1 complex, which are linked to transcription elongation, but how they activate Set1 is not known. Set1 also bears several conserved domains with uncharacterized contributions to activity. Here, we isolated dominant hyperactive SET1(D) alleles, which revealed a complex interplay among Set1 regulatory domains. Remarkably, the RNA-recognition motif (RRM) of Set1 is required for H3K4 trimethylation, but not dimethylation. Also, a central autoinhibitory domain was identified that opposes RRM function by inhibiting trimethylation. Furthermore, a G990E replacement in the catalytic domain conferred Set1 hyperactivity and restored trimethylation to a Set1 derivative bearing mutations in the RRM domain. Surprisingly, certain SET1(D) alleles also partially restored trimethylation to strains lacking histone H2B ubiquitylation or Paf1. Taken together, our data suggest that the catalytic domain of Set1 integrates opposing inputs from the RRM and autoinhibitory domains to link properly H3K4 methylation to the transcript elongation process.
