Methylation profiles of MCF-10A cells infected with MBD2 lentivirus
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE47857 | GEO | 2013/06/13
SECONDARY ACCESSION(S): PRJNA208364
REPOSITORIES: GEO
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Project description:Methylated DNA binding protein 2 (MBD2) has been shown to bind specific methylated promoters and suppress transcription. Here we systematically investigate MBD2 suppression by overexpressing MBD2 in MCF-10A cells and generating genome-wide promoter methylation profiles in order to identify methylation changes likely to affect gene expression levels. MCF-10A cells were infected with MBD2 lentivirus in order to increase MBD2 expression. DNA was extracted from both infected and non-infected cells and enriched for methylated DNA through the method of methylated DNA immunoprecipitation (MeDIP). Enriched and total DNA was hybridized in different channels to 244K custom microarrays (Agilent Technologies) that contained probes covering at 100bp-spacing all transcription start sites from -800bp to 200bp (human, Ensembl v44) and all microRNAs in miRBase from 250bp before to 250bp after the microRNA. Three technical replicates were hybridized for infected and non-infected cells.
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Project description:Methylated DNA binding protein 2 (MBD2) has been shown to bind specific methylated promoters and suppress transcription. Here we systematically investigate MBD2 suppression by overexpressing MBD2 in MCF-10A cells and generating gene expression profiles of overexpressing cells and normal MCF-10A cells.
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Project description:Methylated DNA binding protein 2 (MBD2) has been shown to bind specific methylated promoters and suppress transcription. Here we systematically investigate MBD2 suppression by overexpressing MBD2 in MCF-10A cells and generating gene expression profiles of overexpressing cells and normal MCF-10A cells. MCF-10A cells were infected with MBD2 lentivirus in order to increase MBD2 expression. Total RNA was extracted from both infected and non-infected cells and hybridized to Affymetrix gene expression microarrays. Three technical replicates were hybridized for infected and non-infected cells.
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Project description:We show that in vivo MBD2 is mainly recruited to CpG island promoters that are highly methylated. We also report that MBD2 binds to a subset of CpG island promoters that are characterized by the presence of active histone marks and RNA polymerase II (Pol2). At such sites, MBD2 binds downstream of the transcription start site. Active promoters bound by MBD2 show low to medium gene expression levels and H3K36me3 deposition suggesting a putative role for MBD2 in blocking polymerase II (Pol2) elongation at these promoters. To gain further insight into the function of and epigenetic regulation by MBD2 we generated a tagged version of the protein and stably expressed it in the MCF-7 cell line. We mapped genome wide binding of MBD2 by ChIP sequencing (ChIP-seq) and together with base resolution whole genome bisulfite sequencing (WGBS) we were able to determine the methylation content and the role of methylation density at MBD2 enriched regions. We further dissected MBD2 binding properties, taking advantage of a large set of ChIP-seq data including active histone marks, RNA polymerase II (POL2) and strand specific RNA-seq.
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Project description:We have compared the proteome, transcriptome and metabolome of two isogenic cell lines: MCF-10A, derived from human breast epithelium, and the mutant MCF-10A-H1047R. These cell lines differ by a single amino acid substitution (H1047R) caused by single nucleotide change in one allele of the PIK3CA gene which encodes the catalytic subunit p110α of phosphatidylinositol 3-kinase (PI3K). The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse; with examples in structural protein levels, the DNA repair machinery and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer. 2 cell lines (H1047R and WT), 4 time points (0, 6, 12, 24 hours), 3 replicates
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