Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from Saccharomyces cerevisiae with Rnase H overexpression

ABSTRACT: Transcripts up- or down-regulated comparing a strain where Rnase H is ectopically overexpressed versus wild type (empty vector); assessing the effect of DNA:RNA hybrid degradation on the transcriptome. Strains harboring the RNase H1 over-expression plasmid (p425-GPD-RNase H1) or control plasmid (p425-GPD) were grown in SC-Leucine at 30M-BM-0C. For both sets of microarray experiments, duplicate cultures were analyzed. Total RNA was isolated from 1 unit of A600 mid-log phase cells using a RiboPure Yeast kit (A&B Applied Biosystems), amplified, labeled and fragmented using a Message Apm III RNA Amplification Kit (A&B Applied Biosystems) and hybridized to a GeneChIP Yeast Genome 2.0 array using the GeneChip Hybridization, Wash, and Stain Kit (Affymetrix). Arrays were scanned by the Gene Chip Scanner 3000 7G and expression data was extracted using Expression ConsoleM-bM-^DM-" Software (Affymetrix) with the MAS5.0 statistical algorithm.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Yujia Alina Chan

PROVIDER: E-GEOD-46652 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.

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Expression data from Saccharomyces cerevisiae with Rnase H overexpression

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