Project description:RNA-sequencing of mRNA isolated from in vitro decidualizaing human endometrial stromal cells with or without siRNA-mediated knockdown of steroid receptor coactivator-2/nuclear receptor coactivator 2 (SRC-2/NCOA2)
Project description:Steroid Receptor Coactivator-3 (SRC-3) knockdown in human endometrial stromal cells (HESCs) blocks their decidualization. This result provides translational support for recent studies in the mouse in which conditional SRC-3 knockout in progesterone receptor-positive cells of the endometrium results in early pregnancy loss due to a defect in normal decidualization. RNAseq was performed on the telomerase-immortalised endometrial stromal cell line T-HESC (CRL-4003; American Type Culture Collection) with or without SRC-3 knockdown to identify the transcriptome that is dependent on SRC-3 prior to hormone-dependent HESC decidualization.
Project description:Ovarian estrogen (E2) and progesterone (P4) are indispensable for embryo-implantation and endometrial stromal decidualization; however, the molecular mechanisms that underpin these reproductive processes are unclear. Steroid receptor coregulator-2 (SRC-2) belongs to the multifunctional SRC/p160 family which also includes SRC-1 and SRC-3. Sharing strong sequence homology, all three SRCs exert diverse regulatory effects by modulating the transcriptional potency of nuclear receptor family members, including the estrogen and progesterone receptor (ER and PR respectively). Importantly, absence of SRC-2 in PR positive cells in the epithelial, stromal, and myometrial compartments of the murine uterus results in a striking infertility defect. This reproductive phenotype highlights a key role for SRC-2 in uterine function which is not shared with other coregulators. Intriguingly, abrogation of uterine SRC-2 does not block embryo apposition or attachment to the apical surface of luminal epithelial cells of the endometrium but rather prevents P4-dependent local decidualization of the sub-epithelial stroma. Remarkably, epithelial-specific ablation of SRC-2 in the murine uterus does not compromise endometrial functionality, again underscoring the unique importance of stromal derived SRC-2 in uterine function. The stromal decidualization defect resulting from SRC-2 ablation is reflected at the molecular level by a marked attenuation in P4 responsive target genes known to be critical for P4 dependent decidualization (i.e. ERBB receptor feedback inhibitor 1, Follistatin and Fkbp5). Conversely, the induction of E2 or P4 target genes involved in embryo implantation (i.e. leukemia inhibitory factor (LIF) and Indian hedgehog (Ihh) respectively) is not affected by SRC-2’s absence. As with mouse studies, decidualization of primary human stromal cells (HESCs) in culture is blocked by SRC-2 knockdown; however, HESC decidualization is unaffected by knockdown of SRC-1 or SRC-3. As a consequence of SRC-2 knockdown, molecular studies disclose a striking decrease in the induction of a subset of P4 target genes (i.e. WNT4 and FKBP5) which are essential for the stromal-epithelioid transformation step, the cellular hallmark of endometrial decidualization. Collectively, these studies not only showcase the evolutionary importance of SRC-2 in endometrial biology but also suggest that deregulation of this coregulator may underpin a spectrum of hormone-dependent uterine pathologies such as endometriosis and endometrial cancer. Microarray analysis was performed on mouse uteri using eighteen SRC-2flox/flox (SRC-2f/f) and eighteen PRCre/+ SRC-2flox/flox (SRC-2d/d) mice. Mice were ovariectomized at 6 weeks and after 2 weeks mice were either treated with sesame oil (vehicle) or 1 mg of P4. RNA from three mice per genotype per treatment were pooled and assigned as one sample (three samples per genotype per treatment). multiple group comparison
Project description:Ovarian estrogen (E2) and progesterone (P4) are indispensable for embryo-implantation and endometrial stromal decidualization; however, the molecular mechanisms that underpin these reproductive processes are unclear. Steroid receptor coregulator-2 (SRC-2) belongs to the multifunctional SRC/p160 family which also includes SRC-1 and SRC-3. Sharing strong sequence homology, all three SRCs exert diverse regulatory effects by modulating the transcriptional potency of nuclear receptor family members, including the estrogen and progesterone receptor (ER and PR respectively). Importantly, absence of SRC-2 in PR positive cells in the epithelial, stromal, and myometrial compartments of the murine uterus results in a striking infertility defect. This reproductive phenotype highlights a key role for SRC-2 in uterine function which is not shared with other coregulators. Intriguingly, abrogation of uterine SRC-2 does not block embryo apposition or attachment to the apical surface of luminal epithelial cells of the endometrium but rather prevents P4-dependent local decidualization of the sub-epithelial stroma. Remarkably, epithelial-specific ablation of SRC-2 in the murine uterus does not compromise endometrial functionality, again underscoring the unique importance of stromal derived SRC-2 in uterine function. The stromal decidualization defect resulting from SRC-2 ablation is reflected at the molecular level by a marked attenuation in P4 responsive target genes known to be critical for P4 dependent decidualization (i.e. ERBB receptor feedback inhibitor 1, Follistatin and Fkbp5). Conversely, the induction of E2 or P4 target genes involved in embryo implantation (i.e. leukemia inhibitory factor (LIF) and Indian hedgehog (Ihh) respectively) is not affected by SRC-2’s absence. As with mouse studies, decidualization of primary human stromal cells (HESCs) in culture is blocked by SRC-2 knockdown; however, HESC decidualization is unaffected by knockdown of SRC-1 or SRC-3. As a consequence of SRC-2 knockdown, molecular studies disclose a striking decrease in the induction of a subset of P4 target genes (i.e. WNT4 and FKBP5) which are essential for the stromal-epithelioid transformation step, the cellular hallmark of endometrial decidualization. Collectively, these studies not only showcase the evolutionary importance of SRC-2 in endometrial biology but also suggest that deregulation of this coregulator may underpin a spectrum of hormone-dependent uterine pathologies such as endometriosis and endometrial cancer.
Project description:Endometrial estrogen receptor-α (ESR1) is indispensable for epithelial and stromal proliferation and differentiation during decidualization, yet the gene targets of estradiol (E2) / ESR1 in human stromal cells and associated mechanisms remain unknown. In this study, we characterized global E2-ESR1‒dependent transcriptomic changes and ESR1 recruitment to chromatin. Human endometrial stromal cells were isolated from 4 premenopausal women for primary cell culture. Genome-wide RNA expression by RNA-sequencing was compared in endometrial stromal cells with or without siRNA knockdown of ESR1 in the presence or absence of E2 (n=2). Genome-wide recruitment of ESR1 to chromatin was assessed by chromatin immunoprecipitation sequencing using an antibody against ESR1 was performed to examine binding to target genes (n=1).
Project description:Endometrial estrogen receptor-α (ESR1) is indispensable for epithelial and stromal proliferation and differentiation during decidualization, yet the gene targets of estradiol (E2) / ESR1 in human stromal cells and associated mechanisms remain unknown. In this study, we characterized global E2-ESR1‒dependent transcriptomic changes and ESR1 recruitment to chromatin. Human endometrial stromal cells were isolated from 4 premenopausal women for primary cell culture. Genome-wide RNA expression by RNA-sequencing was compared in endometrial stromal cells with or without siRNA knockdown of ESR1 in the presence or absence of E2 (n=2). Genome-wide recruitment of ESR1 to chromatin was assessed by chromatin immunoprecipitation sequencing using an antibody against ESR1 was performed to examine binding to target genes (n=1).
Project description:We sequenced mRNA of endometrial stromal fibroblasts from six mammalian species. Examination of mRNA levels in endometrial stromal fibroblasts from six mammalian species grown in culture with two biological replicates for each species
Project description:ChickenM-BM- ovalbumin upstream promoter-transcription factor II (COUP-TFII; NR2F2) is an orphan nuclear receptor involved in cell-fate specification, organogenesis, angiogenesis and metabolism. Ablation of COUP-TFII in the mouse uterus causes infertility due to defects in embryo attachment and impaired uterine stromal cell decidualization. Although the function of COUP-TFII in uterine decidualization has been described in mice, its role in the human uterus remains unknown. To better elucidate the mechanisms with which COUP-TFII regulates target gene transcription, genome-wide COUP-TFII binding sites in human endometrial stromal cells (HESC) treated with deciduogenic hormones were identified using ChIP-seq. A total of 16,298 intervals (binding regions) for COUP-TFII were identified compared with the input in HESC chromatin with a very low false discovery rate (0.17%) using a stringent cutoff of p =1x10-10. Distribution of intervals showed that more than half (58.6%) of the COUP-TFII binding sites are located within 10 kb of gene boundaries. 7.5% of total intervals reside within the 10 kb promoter region. A total of 6,077 unique genes were identified to have COUP-TFII binding sites within 10 kb of their gene boundaries. Examination of NR2F2 binding in pooled primary human endometrial stromal cells from 6 healthy women upon decidualization with a hormone cocktail of cAMP, E2 and medroxyprogesterone acetate.