Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Control of chromatin structure by Spt6 has different consequences in coding and regulatory regions

ABSTRACT: Spt6 is a highly conserved factor that carries out important functions in transcription and chromatin structure. To gain new insights into Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant and found that the level of nucleosomes is greatly reduced accross some but not all coding regions. In addition, genome-wide location analyses of RNA polymerase II showed that the nucleosome loss in the spt6 mutant occurs over highly-transcribed genes. Unexpectedly, the effects of the spt6 mutation on nucleosome levels did not correlate with its effects on mRNA levels, suggesting that Spt6 plays distinct roles in controlling chromatin structure across coding regions and in transcriptional regulation. We studied one case of transcriptional regulation by Stp6, at the CHA1 gene, and showed that regulation likely occurs by Spt6 controlling the position of the +1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve and independent function. In order to examine the genome-wide localization of RNAPII and Spt6 in Saccharomyces cerevisiae, RNAPII and Spt6 along with associated DNA sequences were immunoprecipitated using anti-8WG16 and anti-HA antibodies, respectively. The RNAPII and Spt6 chromatin immunoprecipitation was performed in duplicate from WT cells as described below. The extracted DNA was hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome. The combined datasets are available in the supplemental files of the related publication.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: François Robert

PROVIDER: E-GEOD-21787 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Control of chromatin structure by spt6: different consequences in coding and regulatory regions.

Ivanovska Iva I Jacques Pierre-Étienne PÉ Rando Oliver J OJ Robert François F Winston Fred F

Molecular and cellular biology 20101122 3

Spt6 is a highly conserved factor required for normal transcription and chromatin structure. To gain new insights into the roles of Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant. We found that the level of nucleosomes is greatly reduced across some, but not all, coding regions in an spt6 mutant, with nucleosome loss preferentially occurring over highly transcribed genes. This result provides strong support for recent studies that have sugg ...[more]

PMID: 21098123

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Project description:H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of H2A.Z eviction, coupled to its pervasive incorporation by mislocalized SWR-C, alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z removal is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. We profiled H2A.Z occupancy in S. cerevisiae by ChIP-chip on tiling arrays in different mutants for chromatin remodelers and histone chaperones. In most experiments, H2A.Z ChIP samples (Cy5-labeled) were performed using a polyclonal antibody against H2A.Z and hybridized against H2B ChIP samples (Cy3-labeled), also performed using a polyclonal antibody. Temperature-sensitive mutants for the histone chaperones Spt16 and Spt6 (spt16-197 and spt6-1004 respectively) showed strong H2A.Z localization defects so the role of these factors in H2A.Z localization was further analyzed. H2A.Z ChIP-chip experiments in spt16-197 and spt6-1004 were repeated using different experimental designs [normalized against Input DNA or Mock (IgG) ChIP samples]. The contribution of Spt16 and Spt6 on H2A.Z localization was also confirmed by nuclear depletion of Spt16 and Spt6 using the anchor-away system. H2A.Z ChIP-chip experiments were also performed in sic1Δ and spt16-197/sic1Δ cells in order to rule out any G1-arrest artifact. H2A.Z ChIP-chip experiments were repeated using spike-in controls for normalization, revealing widespread H2A.Z occupancy in Spt16 and Spt6 mutants. In addition, the localization of the chromatin remodeler SWR-C was determined in wild type cells as well as in spt16-197 and spt6-1004 cells. Finally, we also profiled histone H4 occupancy by ChIP-chip in wild type, spt16-197, spt6-1004, spt16-197/htz1Δ and spt6-1004/htz1Δ cells. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

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Dataset Information

Control of chromatin structure by Spt6 has different consequences in coding and regulatory regions

Publications

Control of chromatin structure by spt6: different consequences in coding and regulatory regions.

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