Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of aneuploidy by comparative genome hybridization on microarrays

ABSTRACT: Genomic DNA was collected from ies6d or ino80d after undergoing 40 or 80 generations post-sporulation of IES6/ies6 or 20 or 40 generations post-sporulation of INO80/ino80 heterozygous diploid strains, respectively. (1 day = 10 generations.) Genomic DNA was then amplified, and hybridized to a whole genome tiling microarray. Copy number variation was measured relative to a G1 arrested wild type sample.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Grant Brown

PROVIDER: E-MEXP-3576 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification within transcribed regions, but it remains uncertain how these modifications influence, or are influenced by, properties of the elongation complex. Here we establish an intimate link between Cdk9, the kinase component of positive transcription elongation factor b (P-TEFb), and mono-ubiquitylation of histone H2B (H2Bub1), in the fission yeast Schizosaccharomyces pombe. Mutations that impair Cdk9 function reduce H2Bub1 levels in vivo. Conversely, mutations that prevent H2Bub1 decrease phosphorylation of elongation factor subunit Spt5, a sensitive and specific indicator of Cdk9 activity. Chromatin immunoprecipitation (ChIP) analysis suggests this is due to impaired Cdk9 recruitment to H2Bub1-deficient chromatin. P-TEFb and H2Bub1 pathways also interact genetically: mutation of the histone H2B ubiquitin-acceptor residue decreases the requirement for Cdk9 activity in vivo, and multiple cdk9 mutations suppress morphological defects of H2Bub1-deficient strains. Moreover, H2Bub1 loss causes redistribution of transcribing RNAPII on chromatin that is corrected by a hypomorphic cdk9 mutation. Therefore, whereas mutual dependence of Spt5 phosphorylation and H2Bub1 suggests positive feedback between P-TEFb and the ubiquitylation machinery, mutual suppression by cdk9 and H2Bub1-pathway mutations indicates an antagonistic relationship, whereby the activities must be balanced to properly regulate elongation. In order to study the genome-wide localization of H2Bub1 in Schizosaccharomyces pombe, H2Bub1, H2B-Flag as well as RNAPII (along with associated DNA sequences) were immunoprecipitated using repectively anti-H2Bub1, anti-Flag and anti-8WG16 antibodies. The ChIPs were performed in duplicate from WT cells as well as in the H2B-K119R mutant. 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.

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Analysis of aneuploidy by comparative genome hybridization on microarrays

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