Unknown,Transcriptomics,Genomics,Proteomics

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Integrative genomics of gene regulation by estrogen receptors and and coregulators [ChIP-seq]

ABSTRACT: The closely related transcription factors (TFs), estrogen receptors ER? and ER?, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing MCF-7 breast cancer cells with ER?, ER?, or both receptors as a model system to define the basis of differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and transcriptome analyses and use of clustering algorithms delineated 11 clusters representing different chromatin-bound receptor and coregulator assemblies that could be functionally associated through enrichment analysis with distinct patterns of gene regulation and preferential coregulator usage, RIP140 with ER? and SRC3 with ER?. The receptors modified each other’s transcriptional effect, and ER? countered the proliferative drive of ER? through several novel mechanisms associated with specific binding site clusters. Our findings delineate distinct TF-coregulator assemblies that function as control nodes specifying precise patterns of gene regulation, proliferation, and metabolism, as exemplified by two of the most important nuclear hormone receptors in human breast cancer. Examination of Estrogen Receptors alpha and beta and their coregulators SRC3 and RIP140 in different cell types

ORGANISM(S): Homo sapiens

SUBMITTER: Zeynep Madak Erdogan

PROVIDER: E-GEOD-42348 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Integrative genomics of gene and metabolic regulation by estrogen receptors α and β, and their coregulators.

Madak-Erdogan Zeynep Z Charn Tze-Howe TH Jiang Yan Y Liu Edison T ET Katzenellenbogen John A JA Katzenellenbogen Benita S BS

Molecular systems biology 20130618

The closely related transcription factors (TFs), estrogen receptors ERα and ERβ, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing breast cancer cells with ERα, ERβ, or both receptors as a model system to define the basis for differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and ...[more]

PMID: 23774759

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Project description:The closely related transcription factors (TFs), estrogen receptors ERα and ERβ, regulate divergent gene expression programs and proliferative outcomes in breast cancer. Utilizing MCF-7 breast cancer cells with ERα, ERβ, or both receptors as a model system to define the basis of differing response specification by related TFs, we show that these TFs and their key coregulators, SRC3 and RIP140, generate overlapping as well as unique chromatin-binding and transcription-regulating modules. Cistrome and transcriptome analyses and use of clustering algorithms delineated 11 clusters representing different chromatin-bound receptor and coregulator assemblies that could be functionally associated through enrichment analysis with distinct patterns of gene regulation and preferential coregulator usage, RIP140 with ERβ and SRC3 with ERα. The receptors modified each other’s transcriptional effect, and ERβ countered the proliferative drive of ERα through several novel mechanisms associated with specific binding site clusters. Our findings delineate distinct TF-coregulator assemblies that function as control nodes specifying precise patterns of gene regulation, proliferation, and metabolism, as exemplified by two of the most important nuclear hormone receptors in human breast cancer. 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 50. ERβ only cells were generated from these cells by knockdown of ERα in parental cells using the following siERα sequences from Dharmacon: forward, 5’-UCAUCGCAUUCCUUGCAAAdTdT-3’, and reverse, 5’-UUUGCAAGGAAUGCGAUGAdTdT-3’. siRNA experiments were performed as previously described, and resulted in knocknown of ERα mRNA and protein by greater than 95% (GSE4006, PMID 16809442). Briefly, cells were transfected with 20 nM siCtrl [GSE4006] or siERα for 48 h after infection. Then cells were treated with 0.1% EtOH (Veh) or 10 nM E2 (Sigma-Aldrich) for 24h. All experiments were conducted with two replicates. key words; Adenovirus infection,siRNA knock-down, ligand treatment

Project description:Cell-and context-specific activities of nuclear receptors may in part be due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We have shown previously that silencing of RIP140 enhances RA-induced differentiation and enhances the induction of model RA target genes in human embryonal carcinoma cells (EC). Through use of microarray technology we sought to elucidate in a de novo fashion the global role of RIP140 in RA-dependent signaling. RIP140-dependent gene expression was largely consistent with RIP140 functioning to limit RAR signaling. Few if any genes were regulated in a manner to support a role for RIP140 in â??active repressionâ??. Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140, suggesting that RIP140 may discriminate between different classes of RA target genes. RIP140 silencing also accelerated RA target gene activation and sensitized EC cells to low doses of RA. Together the data suggests that the RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation. RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. Experiment Overall Design: The current investigation sought to determine the global effects of RIP140 on RA-dependent and RAâ??independent gene expression. A dose of siRNA of 90 nM was chosen. Twelve independent hybridizations were performed from mRNA isolated from 12 independent samples. One scrambled and two independent RISC-free siRNA transfections and transfections with three distinct RIP140 siRNAs were performed in NT2/D1 cells. Each siRNA treatment was further treated with either 10 Î¼M RA or DMSO control for 24 hours. Hence, the design consisted of four groups, the three siRNA controls treated with vehicle, Group 1; the siRNA controls treated with RA, Group 2; siRNA to RIP140 treated with vehicle, Group 3; and siRNA to RIP140 treated with RA, Group 4 . Experiment Overall Design: The treatments were done in the presence of normal sera, since charcoal absorption of sera removes many small hydrophobic ligands to the nuclear receptor family in addition to retinoids. This measure was taken with the aim to perhaps identify target genes of other nuclear receptors including orphan receptors that could be regulated by RIP140 in the presence of low levels of their cognate ligands. RA treatment of 10 Î¼M for 24 hours was chosen since previous experience with NT2/D1 cells indicated that a reasonably sized set of altered genes would be obtained. In addition, NT2/D1 cells commit to RA-induced differentiation by 48 hours and RIP140 repression enhances and accelerates the effects of RA in these cells. Therefore, the 24-hour time point is well-within the range of the RA commitment â??windowâ?? and a larger percentage of direct RA-target genes could be expect to be uncovered compared to later time points. Experiment Overall Design: Based on the current understanding of the properties of RIP140, a number of potential outcomes were anticipated: 1) RIP140 may limit RA target gene activation by negative-feedback inhibition upon RA addition, 2) RIP140 may participate in â??active repressionâ?? of gene expression upon RA addition, 3) an unexpected outcome that RIP140 may participate in the activation of a subset of target genes, 4) RIP140 may regulate the transcription of target genes of other nuclear receptors or even non-nuclear receptor transcription factors. Experiment Overall Design: Total RNA was purified using RNeasy columns (Qiagen). Hybridizations were performed according to Affymetrix guidelines at the Dartmouth College Microarray Shared Resource using an Affymetrix GeneChip Workstation. Biotin-labeled cRNA was generated from 5 Î¼g of total RNA and hybridized to the Human Genome (HG) U133 Plus 2.0 chip A total of 12 hybridizations were performed comprising 12-independent biologic samples organized into the four groups of three.

Dataset Information

Integrative genomics of gene regulation by estrogen receptors and and coregulators [ChIP-seq]

Publications

Integrative genomics of gene and metabolic regulation by estrogen receptors α and β, and their coregulators.

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