Dataset Information

Identification of histone mutants that are defective for transcription-coupled nucleosome occupancy.

ABSTRACT: Our previous studies of Saccharomyces cerevisiae described a gene repression mechanism where the transcription of intergenic noncoding DNA (ncDNA) (SRG1) assembles nucleosomes across the promoter of the adjacent SER3 gene that interfere with the binding of transcription factors. To investigate the role of histones in this mechanism, we screened a comprehensive library of histone H3 and H4 mutants for those that derepress SER3. We identified mutations altering eight histone residues (H3 residues V46, R49, V117, Q120, and K122 and H4 residues R36, I46, and S47) that strongly increase SER3 expression without reducing the transcription of the intergenic SRG1 ncDNA. We detected reduced nucleosome occupancy across SRG1 in these mutants to degrees that correlate well with the level of SER3 derepression. The histone chromatin immunoprecipitation experiments on several other genes suggest that the loss of nucleosomes in these mutants is specific to highly transcribed regions. Interestingly, two of these histone mutants, H3 R49A and H3 V46A, reduce Set2-dependent methylation of lysine 36 of histone H3 and allow transcription initiation from cryptic intragenic promoters. Taken together, our data identify a new class of histone mutants that is defective for transcription-dependent nucleosome occupancy.

SUBMITTER: Hainer SJ

PROVIDER: S-EPMC3165560 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Identification of histone mutants that are defective for transcription-coupled nucleosome occupancy.

Hainer Sarah J SJ Martens Joseph A JA

Molecular and cellular biology 20110705 17

Our previous studies of Saccharomyces cerevisiae described a gene repression mechanism where the transcription of intergenic noncoding DNA (ncDNA) (SRG1) assembles nucleosomes across the promoter of the adjacent SER3 gene that interfere with the binding of transcription factors. To investigate the role of histones in this mechanism, we screened a comprehensive library of histone H3 and H4 mutants for those that derepress SER3. We identified mutations altering eight histone residues (H3 residues ...[more]

PMID: 21730290

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Project description:The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome "remodeler" rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4's action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription-one that requires the action of SWI/SNF recruited by the activator, and a slower one that does not-clarifies our understanding of the early events of gene activation, and in particular corrects earlier reports that SWI/SNF plays no role in GAL gene induction. Our finding that chromatin structure is irrelevant for repression as studied here-that is, repression sets in as efficiently whether or not promoter nucleosomes are allowed to reform-contradicts the widely held, but little tested, idea that nucleosomes are required for repression. These findings were made possible by our nucleosome occupancy assay. The assay, we believe, will prove useful in studying other outstanding issues in the field.

Project description:Previous studies in Saccharomyces cerevisiae established that depletion of histone H4 results in the genome-wide transcriptional de-repression of hundreds of genes. To probe the mechanism of this transcriptional de-repression, we depleted nucleosomes in vivo by conditional repression of histone H3 transcription. We then measured the resulting changes in transcription by RNA-seq and in chromatin organization by MNase-seq. This experiment also bears on the degree to which trans-acting factors and DNA-encoded elements affect nucleosome position and occupancy in vivo. We identified ?60,000 nucleosomes genome wide, and we classified ?2,000 as having preferentially reduced occupancy following H3 depletion and ?350 as being preferentially retained. We found that the in vivo influence of DNA sequences that favor or disfavor nucleosome occupancy increases following histone H3 depletion, demonstrating that nucleosome density contributes to moderating the influence of DNA sequence on nucleosome formation in vivo. To identify factors important for influencing nucleosome occupancy and position, we compared our data to 40 existing whole-genome data sets. Factors associated with promoters, such as histone acetylation and H2A.z incorporation, were enriched at sites of nucleosome loss. Nucleosome retention was linked to stabilizing marks such as H3K36me2. Notably, the chromatin remodeler Isw2 was uniquely associated with retained occupancy and altered positioning, consistent with Isw2 stabilizing histone-DNA contacts and centering nucleosomes on available DNA in vivo. RNA-seq revealed a greater number of de-repressed genes (?2,500) than previous studies, and these genes exhibited reduced nucleosome occupancy in their promoters. In summary, we identify factors likely to influence nucleosome stability under normal growth conditions and the specific genomic locations at which they act. We find that DNA-encoded nucleosome stability and chromatin composition dictate which nucleosomes will be lost under conditions of limiting histone protein and that this, in turn, governs which genes are susceptible to a loss of regulatory fidelity.

Dataset Information

Identification of histone mutants that are defective for transcription-coupled nucleosome occupancy.

Publications

Identification of histone mutants that are defective for transcription-coupled nucleosome occupancy.

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