Cellular reprogramming by the conjoint action of ERalpha, FOXA1, and GATA3 to a ligand inducible growth state
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE30574 | GEO | 2011/09/05
SECONDARY ACCESSION(S): PRJNA143371
REPOSITORIES: GEO
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Project description:Despite the role of the estrogen receptor alpha (ERalpha) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERalpha into ERalpha-negative cells paradoxically has been growth inhibition. We map the binding profiles of ERalpha and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells. We observe that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERalpha alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 coactivator recruitment, RNA Pol II occupancy, and chromatin opening. The enhancesome binding sites appear to regulate the genes driving core ERalpha function. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERalpha-negative MDA-MB-231 and BT-459 cells to restore the estrogen responsive growth and to transcriptionally resemble the estrogen treated ERalpha-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERalpha, FOXA1 and GATA3 are necessary for the full repertoire of cancer associated effects of the ERalpha. Illumina HumanRef-8 v3 Expression BeadChip was used to measure the gene expression levels from ERalpha negative breast cancer cell MDA-MB-231 with different transfections: with vector control, with ERalpha only, or with ERalpha+FOXA1+GATA3 combined.
2011-09-04 | E-GEOD-30574 | biostudies-arrayexpress
Project description:Despite the role of the estrogen receptor alpha (ERalpha) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERalpha into ERalpha-negative cells paradoxically has been growth inhibition. We map the binding profiles of ERalpha and its interacting transcription factors (TFs), FOXA1 and GATA3, in MCF-7 breast carcinoma cells. We observe that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERalpha alone. We demonstrate that these enhanceosome-occupied sites are associated with optimal enhancer characteristics with highest p300 coactivator recruitment, RNA Pol II occupancy, and chromatin opening. The enhancesome binding sites appear to regulate the genes driving core ERalpha function. Most importantly, we show that transfection of all three TFs was necessary to reprogram the ERalpha-negative MDA-MB-231 and BT-459 cells to restore the estrogen responsive growth and to transcriptionally resemble the estrogen-treated ERalpha-positive MCF-7 cells. Cumulatively, these results suggest that all of the enhanceosome components comprising ERalpha, FOXA1 and GATA3 are necessary for the full repertoire of the cancer-associated effects of the ERalpha. The analysis of ERalpha, FOXA1, and GATA3 in MCF-7 cancer cells was done by ChIP-seq data obtained either with estradiol (E2) stimulation or without stimulation using vehicle as a control. Using the ERalpha bindings defined by ChIP-seq (GSE23893), FOXA1 bindings (GSE26831), and GATA3 bindings (this Series), we analyzed the enhanceosome effect of the overlapped binding sites from ERalpha, FOXA1 and GATA3.
2011-09-05 | E-GEOD-29073 | biostudies-arrayexpress
Project description:Despite the role of the estrogen receptor alpha (ERalpha) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERalpha into ERalpha-negative cells paradoxically has been growth inhibition. We map the binding profiles of ERalpha and its interacting transcription factors (TFs), FOXA1 and GATA3, in MCF-7 breast carcinoma cells. We observe that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERalpha alone. We demonstrate that these enhanceosome-occupied sites are associated with optimal enhancer characteristics with highest p300 coactivator recruitment, RNA Pol II occupancy, and chromatin opening. The enhancesome binding sites appear to regulate the genes driving core ERalpha function. Most importantly, we show that transfection of all three TFs was necessary to reprogram the ERalpha-negative MDA-MB-231 and BT-459 cells to restore the estrogen responsive growth and to transcriptionally resemble the estrogen-treated ERalpha-positive MCF-7 cells. Cumulatively, these results suggest that all of the enhanceosome components comprising ERalpha, FOXA1 and GATA3 are necessary for the full repertoire of the cancer-associated effects of the ERalpha.
2011-09-05 | GSE29073 | GEO
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. Keywords: time course
2008-05-07 | GSE11352 | GEO
Project description:We used ChIP-Seq to map ERalpha binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF-7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identified 10,205 high confidence ERalpha binding sites in response to E2 of which 68% contained an estrogen response element (ERE) and only 7% contained a FOXA1 motif. Remarkably, 596 genes already change significantly in RNAPII occupancy (59% up and 41% down) following one hour of E2 exposure. Although pausing of RNA polymerase II occurs frequently in MCF-7 cells (17%) it is only observed on a minority of E2-regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERalpha DNA binding and prevent RNAPII loading on the promoter and coding body on E2-upregulated genes. Both antagonists act differently on E2-downregulated genes. Tamoxifen acts as an agonist, also downregulating these genes while fulvestrant antagonizes E2 induced repression and often increases RNAPII occupancy. Furthermore our data identified genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus, antagonist loaded ERalpha acts mechanistically different on E2-activated and E2-repressed genes.
2009-03-09 | GSE14664 | GEO
Project description:We used ChIP-Seq to map ERalpha binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF-7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identified 10,205 high confidence ERalpha binding sites in response to E2 of which 68% contained an estrogen response element (ERE) and only 7% contained a FOXA1 motif. Remarkably, 596 genes already change significantly in RNAPII occupancy (59% up and 41% down) following one hour of E2 exposure. Although pausing of RNA polymerase II occurs frequently in MCF-7 cells (17%) it is only observed on a minority of E2-regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERalpha DNA binding and prevent RNAPII loading on the promoter and coding body on E2-upregulated genes. Both antagonists act differently on E2-downregulated genes. Tamoxifen acts as an agonist, also downregulating these genes while fulvestrant antagonizes E2 induced repression and often increases RNAPII occupancy. Furthermore our data identified genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus, antagonist loaded ERalpha acts mechanistically different on E2-activated and E2-repressed genes. Examination of ERalpha binding sites upon the binding of different ligands and association with transcription via RNAPII occupancy.
2009-03-09 | E-GEOD-14664 | 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 manufacturerâs protocols.
2009-01-09 | E-GEOD-11352 | biostudies-arrayexpress
Project description:The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-M-NM-1 (ERM-NM-1)-positive breast tumors in which it participates with ERa and FOXA1 in a complex transcriptional regulatory program driving tumor growth. Paradoxically, GATA3 mutations are frequent in breast cancer and have been classified as drivers. To elucidate the contribution(s) of GATA3 alterations to oncogenesis, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERa and FOXA1 following estrogen stimulation. Thus, mutant GATA3 uncouples protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified stronger accumulation of GATA3 in cells bearing the mutation, albeit with a similar distribution across the genome. We propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of mammary epithelial cells to hormone signaling, thus conferring a selective growth advantage. Genome-wide mapping of GATA3 in two cell lines. There were two biological replicates and unchipped (input) DNA was used as reference.
2014-04-01 | E-GEOD-51274 | biostudies-arrayexpress
Project description:Two subtypes of the estrogen receptor, ERalpha and ERbeta, mediate the actions of estrogens, and the majority of human breast tumors contain both ERalpha and ERbeta. To examine the possible interactions and modulatory effects of ERbeta on ERalpha activity, we have used adenoviral gene delivery to produce human breast cancer (MCF-7) cells expressing ERbeta, along with their endogenous ERalpha. We have examined the effects of ERβ expression on genome-wide gene expression by Affymetrix GeneChip microarrays. We find that ERbeta modulated estrogen gene expression on nearly 24% of E2-stimulated genes but only 8% of E2-inhibited genes. We find that ERbeta modulation is gene-specific, enhancing or counteracting ERalpha regulation for distinct subsets of estrogen target genes. Introduction of ERbeta into ERalpha-containing cells induced up/down-regulation of many estrogen target in the absence of any added ligand. In addition, ERbeta presence elicited the expression of a unique set of genes that were not regulated by ERalpha alone. ERbeta modulated the expression of genes in many functional categories, but the greatest numbers were associated with transcription factor and signal transduction pathways. Regulation of multiple components in the TGF beta, SDF1, and semaphorin pathways, may contribute to the suppression of proliferation observed with ERbeta both in the presence and absence of estrogen. Hence, ERbeta modulates ERalpha gene regulation in diverse ways that may contribute to its growth-inhibiting beneficial effects in breast cancer Experiment Overall Design: MCF-7 cells expressing endogenous ERalpha were infected with adenovirus carrying either estrogen receptor beta (AdERb) or no insert (Ad) at multiplicity of infection (moi) of 5 or 50. Cells were infected with adenovirus for a period of 48hr before treatment with ligand (vehicle control or 10nM 17beta-estradiol) for a additional period of 24hr before harvest.
2009-04-24 | E-GEOD-4006 | biostudies-arrayexpress
Project description:Estrogen Receptor (ER) is a steroid hormone receptor that regulates epithelial genes in breast cancer. ER forms a regulatory network with the other transcription factors, FOXA1 and GATA3. GATA3 is known to be capable of specifying chromatin localization of FOXA1 and ER. GATA3 has been identified as one of the most frequently mutated genes in breast cancer. However, how GATA3 mutations impact this transcriptional network is unknown. Here we investigate the function of one of the recurrent patient-derived GATA3 mutations (R330fs) for this regulatory network. Genomic analysis indicates that the R330fs mutant can disrupt the cooperative action of ER, FOXA1, and GATA3, and induce chromatin relocalization of these factors. Relocalizations of ER and FOXA1 are associated with altered chromatin architectures leading to differential gene expression in the GATA3 mutant cells. These results suggest the active role of GATA3 mutants in ER positive breast tumors.
2020-02-27 | GSE130703 | GEO