Unexpected nuclear hormone receptor and chromatin dynamics regulate estrous cycle dependent gene expression [HIF2A cKO RNA-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE268491 | GEO | 2024/08/01
REPOSITORIES: GEO
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Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
2019-03-21 | PXD012930 | Pride
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
2024-08-01 | GSE234064 | GEO
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
2024-08-01 | GSE234061 | GEO
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
2024-08-01 | GSE234059 | GEO
Project description:Hormone dependent uterine gene expression changes that occur during the estrous cycle suggest hormone receptor binding to chromatin may also be dynamic. Therefore, we employed a multi-faceted approach to examine in vivo dynamics of hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq for estrogen receptor alpha (ERα) and progesterone receptor (PGR). Genome wide, there were extensive estrous cycle dependent changes in ERα and PGR binding as well as chromatin accessibility. There were 4,159 differentially expressed genes between estrus and diestrus. At transcription start sites, accessibility generally correlated with the directionality of gene expression and there was reduced PGR in estrus compared to diestrus but little change in ERα. There were 2,727 enhancers with dynamic accessibility near these genes and 77% of those correlated with directionality of gene expression changes. However, most enhancers were constitutively open (8,694). In both dynamic and constitutively open enhancers, ERα and PgR binding was coordinately lost from diestrus to estrus. Diestrus specific ERα binding and accessible regions were enriched for PGR, FOX, GATA and SOX binding motifs. In contrast, estrus specific ERα binding occurred at transcription factor deserts in relatively closed chromatin while estrus specific accessible regions were highly enriched for ATF, ELF and ELK motifs. Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) revealed many estrous cycle dependent partners of ERα (diestrus, 60; estrus, 24). PGR was found in complex with ERα during diestrus but not estrus supporting coordinated binding of both receptors during diestrus. Two of the cohesin complex proteins, SMC1A and SMC3, were found in complex with ERα during diestrus but not estrus; overlap of SMC1A with ERα confirmed this preferential interaction during diestrus. Additionally, HiC analysis showed more diestrus specific interactions than estrus (476 versus 263) suggesting the SMC1A/ ERα interactions have functional consequences on chromatin structure. Taken together, a complex series of interactions between hormone receptors, chromatin structure and accessibility orchestrate estrous cycle dependent changes in gene expression.
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Project description:Identification of ChIP-seq and RIME grade antibodies for estrogen receptor alpha
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Project description:Methods for identifying protein-protein interactions have mostly been limited to tagged exogenous expression approaches. We now establish a rapid, robust and comprehensive method for finding interacting proteins using endogenous proteins from limited cell numbers. We apply this approach called ‘Rapid IP-Mass Spectrometry of Endogenous proteins (RIME)’ to identify ER, FoxA1 and E2F4 interacting proteins in breast cancer cells. From small numbers of starting cells, we find a comprehensive collection of known ER, FoxA1 and E2F4 targets, plus a number of novel unexpected interactors. One of the most ER (and FoxA1) associated interactors is GREB1, an estrogen induced gene with almost no known function. We apply RIME, in parallel with ER ChIP-seq, to identify ER protein interactors and ER binding events from solid tumor xenografts, resulting in the validation of the ER-GREB1 interactions. Furthermore, we establish a method for identifying endogenous interacting proteins from solid primary breast cancer samples, whih we apply to validate ER interactions with GREB1 and additional co-factors. Mechanistically, we show that GREB1 is recruited with ER to the chromatin where it functions as an essential estrogen-mediated regulatory factor required for effective ER transcriptional activity. Our novel approach enables, for the first time, the ability for discovery and validation of protein-protein interactions in whole tissue and solid tumors, revealing significant insight into ER regulatory factors. Examination of ERGREB1 and E2F4 genomic binding patterns in cell line and xenograft tumour models
2013-01-18 | E-GEOD-41561 | biostudies-arrayexpress
Project description:Methods for identifying protein-protein interactions have mostly been limited to tagged exogenous expression approaches. We now establish a rapid, robust and comprehensive method for finding interacting proteins using endogenous proteins from limited cell numbers. We apply this approach called ‘Rapid IP-Mass Spectrometry of Endogenous proteins (RIME)’ to identify ER, FoxA1 and E2F4 interacting proteins in breast cancer cells. From small numbers of starting cells, we find a comprehensive collection of known ER, FoxA1 and E2F4 targets, plus a number of novel unexpected interactors. One of the most ER (and FoxA1) associated interactors is GREB1, an estrogen induced gene with almost no known function. We apply RIME, in parallel with ER ChIP-seq, to identify ER protein interactors and ER binding events from solid tumor xenografts, resulting in the validation of the ER-GREB1 interactions. Furthermore, we establish a method for identifying endogenous interacting proteins from solid primary breast cancer samples, whih we apply to validate ER interactions with GREB1 and additional co-factors. Mechanistically, we show that GREB1 is recruited with ER to the chromatin where it functions as an essential estrogen-mediated regulatory factor required for effective ER transcriptional activity. Our novel approach enables, for the first time, the ability for discovery and validation of protein-protein interactions in whole tissue and solid tumors, revealing significant insight into ER regulatory factors. Cells were transfected with siControl or siGREB1. The cells were treated with 10nM ICI 182780 for 24 hr to further deplete ER. 48 hr after transfection, the cells were treated with 100nM of Estrogen or vehicle for 6 hr and total RNA was collected from four biological replicates
2013-01-18 | E-GEOD-37386 | biostudies-arrayexpress
Project description:We report the ER alpha regulatory network of Tamoxifen resistance MCF7 cell line using the Chromatin immunoprecipitated high-throughput sequencing technology (ChIP-seq). By Integrating the gene expression data (previously reported) with the ChIP-seq data, we generated ER alpha regulatory network and pathways. For ER alpha regulatory network, hub TFs with enriched motifs were identified from ER alpha peak together with PolII peaks. We then scan the position weight matrix (PWM) of ER alpha peak region of certain gene to find out the regulatory relationship between hub TF and normal TF. For regulatory pathway, genes were grouped base on their expression value at 4 different time point. Then the hub TF that plays important role in each time point of each group was identified. This study provides a framework for the application of ChIP-seq and gene expression data for the construction of ER alpha regulatory network. 4 different ChIP-seq dataset in Tamoxifen resistance MCF7 cell line
2010-12-20 | E-GEOD-26083 | biostudies-arrayexpress
Project description:We report the ER alpha regulatory network of Tamoxifen resistance MCF7 cell line using the Chromatin immunoprecipitated high-throughput sequencing technology (ChIP-seq). By Integrating the gene expression data (previously reported) with the ChIP-seq data, we generated ER alpha regulatory network and pathways. For ER alpha regulatory network, hub TFs with enriched motifs were identified from ER alpha peak together with PolII peaks. We then scan the position weight matrix (PWM) of ER alpha peak region of certain gene to find out the regulatory relationship between hub TF and normal TF. For regulatory pathway, genes were grouped base on their expression value at 4 different time point. Then the hub TF that plays important role in each time point of each group was identified. This study provides a framework for the application of ChIP-seq and gene expression data for the construction of ER alpha regulatory network.
2010-12-20 | GSE26083 | GEO