Analysis of the Phosphorylated Estrogen Receptor-α Cistrome Reveals Association with Direct ER-DNA Binding and the Transcription Factor GRHL2
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ABSTRACT: Phosphorylation of the estrogen receptor-α (ER) at serine 118 (pS118-ER) is involved in modulating ER-dependent gene transcription and recruitment of ER to certain gene promoters. While the effects of pS118 on ER-DNA interactions have been investigated at specific sites, the genome-wide binding profile (cistrome) of pS118-ER remains to be studied. To characterize the pS118-ER cistrome, ChIP-seq was performed on pS118-ER and pan-ER in MCF-7 cells. A subset of ER sites was occupied by pS118-ER and these sites were associated with the active enhancer mark H3K27ac. Additionally, pS118-ER sites were found to be enriched in the DNA binding motif for GRHL2 as well as the estrogen response element relative to all ER sites. Utilizing a DNA binding microarray, pS118-ER was found to be more commonly associated with direct DNA binding events compared to indirect binding events. These results suggest a role for pS118-ER at active enhancers and as a regulator of direct ER-DNA interactions.
Project description:Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant post-translational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group found enrichment of the GRHL2 motif near pS118-ER binding sites. In this report we use cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300. Transcriptomic analysis yielded four subsets of ER/GRHL2 co-regulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis identified several coregulated genes involved in cell migration. Accordingly, knockdown of GRHL2 combined with estrogen treatment resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to select enhancers and facilitates pS118-ER recruitment to chromatin which then results in differential activation and repression of genes that control ER-positive breast cancer cell migration.
Project description:Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant post-translational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group found enrichment of the GRHL2 motif near pS118-ER binding sites. In this report we use cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300. Transcriptomic analysis yielded four subsets of ER/GRHL2 co-regulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis identified several coregulated genes involved in cell migration. Accordingly, knockdown of GRHL2 combined with estrogen treatment resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to select enhancers and facilitates pS118-ER recruitment to chromatin which then results in differential activation and repression of genes that control ER-positive breast cancer cell migration.
Project description:Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant post-translational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group found enrichment of the GRHL2 motif near pS118-ER binding sites. In this report we use cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300. Transcriptomic analysis yielded four subsets of ER/GRHL2 co-regulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis identified several coregulated genes involved in cell migration. Accordingly, knockdown of GRHL2 combined with estrogen treatment resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to select enhancers and facilitates pS118-ER recruitment to chromatin which then results in differential activation and repression of genes that control ER-positive breast cancer cell migration.
Project description:Estrogen Receptor ? (ER?) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ER? has functions which are independent of ligands. In the present work, we investigated the binding of ER? to chromatin in absence of ligands, and its function(s) on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ER? binds to more than four thousands chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ER? binding is specifically linked to genes with developmental functions, as compared to estrogen-induced binding. Moreover, we found that siRNA-mediated downregulation of ER? in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Downregulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ER? downregulation using shRNA, which caused cell-growth arrest, was accompanied by increased H3K27me3 at ER? binding sites. Finally, we found that FOXA1 and AP2? binding to several sites is decreased upon ER? silencing, suggesting that unliganded ER? participates, together with other factors, to the maintenance of the luminal-specific cistrome in breast cancer cells. Examination of unligandend estrogen receptor alpha (apoER?) DNA interactions in control and ER? siRNA treated MCF7 cells.
Project description:Analysis of the Phosphorylated Estrogen Receptor-α Cistrome Reveals Association with Direct ER-DNA Binding and the Transcription Factor GRHL2
Project description:Estrogen-based treatments have numerous extra-reproductive effects. On the one hand, they have protective metabolic actions against abdominal fat accumulation, type 2 diabetes, liver steatosis,⦠But, on the other hand, the oral route of administration, presumably due to a hepatic impact on liver-derived coagulation factors, appears to increase risks of venous thrombosis and pulmonary embolism. The characterization of liver genes expression regulations by these hormones thereby appears of utmost interest, but the estrogen-sensitive transcriptome of liver and ER cistrome was so far explored under chronic hormonal treatment. To explore the early steps of these mechanisms, we determined here the short-term changes in liver transcriptional responses to acute E2 administration, and aimed to characterize the mechanisms allowing these programs to be engaged. Collectively, through the comparison of mice expressing or not ER, our data demonstrate that acute administration of E2 provokes genes expression variations which fit with a crucial role of ER in the prevention of liver steatosis in mice fed with a high fat diet by E2. They also evidenced higher proportion of ER binding sites (BSs) located at the direct vicinity (distance <3kb) of regulated genes than what is determined in human cancer cell models. Besides this specific aspect of ER cistrome in vivo, we unexpectedly found that 40 % of the BSs of the pioneer factor Foxa2 were dependent upon the expression of ER that indirectly influences the distribution of the nucleosomes harbouring a dimethylated H3K4 around these Foxa2 sites. Whole-genome analysis of estrogen receptor (ER) and FOXA2 binding events associated with the cartography of two histone marks (H3K4me2 and H3K27ac) in livers from wild-type or ERKO mice.
Project description:Differential ChIP-Seq data monitoring changes in active enhancer marks (H3K27ac sites) after treatment with siGRHL2 in MCF7 and T47D breast cancer models. Comparing sites altered by treatment with siGRHL2 after 48hours revealed these sites to be enriched for Estrogen Receptor (ER) binding.
Project description:GRHL2 enhances phosphorylated estrogen receptor DNA binding and regulates ER-mediated transcriptional activation and repression[ChIP-seq]