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Transcription profiling of human breast cancer cells expressing ERalpha and treated with 17beta-estradiol

ABSTRACT: Gene expression changes caused by estrogen treatment of breast cancer cells that re-express ERalpha was investigated by infecting ER-negative MDA-MB-231 breast cancer cells for 24 h with recombinant adenovirus encoding full-length human ERalpha (Ad-ERalpha) or control vector (Ad-LacZ), and treating them with 0·01% ethanol (vehicle control) or 10-8 M 17beta-estradiol (E2). After 48 h of treatment, total RNA was isolated and used for transcript profiling on Affymetrix GeneChips. Three independent biological replicates of this experiment were carried out.

ORGANISM(S): Homo sapiens

SUBMITTER: Moggs JG

PROVIDER: S-ECPF-GEOD-2251 | biostudies-other | 2005 Apr

REPOSITORIES: biostudies-other

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Project description:Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation. Experiment Overall Design: We used oligonucleotide expression microarrays (Affymetrix GeneChip U133 Plus 2.0) to identify estradiol (E2)-responsive genes in the estrogen-receptor positive breast cancer cell line, MCF7. MCF7 cells were grown to 30-50% confluency and exposed to 10 nM E2 (or vehicle only) at 12, 24, and 48 hours. Each timepoint was performed in triplicate (ie, biological replicates). Total RNA was isolated from cells using the Qiagen RNeasy kit, and 5 micrograms of total RNA was amplified, labeled and hybridized to the array according to the manufacturer’s protocols.

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Transcription profiling of human breast cancer cells expressing ERalpha and treated with 17beta-estradiol

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