Transcription profiling time series of human estrogen-responsive human breast cancer cells treated with 17 beta-estradiol
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Mutarelli M
PROVIDER: S-ECPF-TABM-631 | biostudies-other | 2008
REPOSITORIES: biostudies-other
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Project description:Human estrogen-responsive breast cancer cell line ZR-75-1 was treated with 10nM of 17 beta-estradiol. Gene expression was analyzed on total RNA extracted before or after 1, 2, 4, 6, 8, 12, 16, 20, 24, 28 and 32 hours hormonal stimulation. For each condition 2 technical replicates were performed, except the reference sample (before stimulation - 0h) which was in quadruplicate and the 4h sample in triplicate.
2009-02-03 | E-TABM-631 | biostudies-arrayexpress
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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Project description:Human estrogen-responsive breast cancer cell lines ZR-75-1 and MCF-7 were treated with 10nM of 17beta-estradiol. Gene expression was analyzed on total RNA extracted before or after 1, 2, 4, 6, 8, 12, 16, 20, 24, 28 and 32 hours hormonal stimulation. For each condition 2 technical replicates were performed, except the reference sample (before stimulation - 0h) which was in respectively in quadruplicate and quintuplicate.
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Project description:The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation,particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer. Experiment Overall Design: This Series currently contains the gene expression data accompaning Carroll JS et al. Nature Genetics 38,1289-1297(2006). MCF7 cells were stimulated with 100 nM estrogen for 0, 3, 6, or 12h. All experiments were performed in triplicate.
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