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Estrogen receptor α (ERα) modulates gene expression by interacting with chromatin regions that are frequently distal from the promoters of estrogen-regulated genes. Active chromatin-enriched "super-enhancer" (SE) regions, mainly observed in in vitro culture systems, often control production of key cell type-determining transcription factors. Here, we defined super-enhancers that bind to ERα in vivo within hormone-responsive uterine tissue in mice. We found that SEs are already formed prior to estrogen exposure at the onset of puberty. The genes at SEs encoded critical developmental factors, including retinoic acid receptor α (RARA) and homeobox D (HOXD). Using high-throughput chromosome conformation capture (Hi-C) along with DNA sequence analysis, we demonstrate that most SEs are located at a chromatin loop end and that most uterine genes in loop ends associated with these SEs are regulated by estrogen. Although the SEs were formed before puberty, SE-associated genes acquired optimal ERα-dependent expression after reproductive maturity, indicating that pubertal processes that occur after SE assembly and ERα binding are needed for gene responses. Genes associated with these SEs affected key estrogen-mediated uterine functions, including transforming growth factor β (TGFβ) and LIF interleukin-6 family cytokine (LIF) signaling pathways. To the best of our knowledge, this is the first identification of SE interactions that underlie hormonal regulation of genes in uterine tissue and optimal development of estrogen responses in this tissue., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Insulin-like growth factor 1 (IGF1) is primarily synthesized in and secreted from the liver; however, estrogen (E2), through E2 receptor α (ERα), increases uterine Igf1 mRNA levels. Previous ChIP-seq analyses of the murine uterus have revealed a potential enhancer region distal from the Igf1 transcription start site (TSS) with multiple E2-dependent ERα-binding regions. Here, we show E2-dependent super enhancer-associated characteristics and suggest contact between the distal enhancer and the Igf1 TSS. We hypothesized that this distal super-enhancer region controls E2-responsive induction of uterine Igf1 transcripts. We deleted 430 bp, encompassing one of the ERα-binding sites, thereby disrupting interactions of the enhancer with gene-regulatory factors. As a result, E2-mediated induction of mouse uterine Igf1 mRNA is completely eliminated, whereas hepatic Igf1 expression remains unaffected. This highlights the central role of a distal enhancer in the assembly of the factors necessary for E2-dependent interaction with the Igf1 TSS and induction of uterus-specific Igf1 transcription. Of note, loss of the enhancer did not affect fertility or uterine growth responses. Deletion of uterine Igf1 in a PgrCre;Igf1f/f model decreased female fertility but did not impact the E2-induced uterine growth response. Moreover, E2-dependent activation of uterine IGF1 signaling was not impaired by disrupting the distal enhancer or by deleting the coding transcript. This indicated a role for systemic IGF1, suggested that other growth mediators drive uterine response to E2, and suggested that uterine-derived IGF1 is essential for reproductive success. Our findings elucidate the role of a super enhancer in Igf1 regulation and uterine growth.

Estrogen receptor α (ERα) action plays an important role in pancreatic β-cell function and survival; thus, it is considered a potential therapeutic target for the treatment of type 2 diabetes in women. However, the mechanisms underlying the protective effects of ERα remain unclear. Because ERα regulates mitochondrial metabolism in other cell types, we hypothesized that ERα may act to preserve insulin secretion and promote β-cell survival by regulating mitochondrial-endoplasmic reticulum (EndoRetic) function. We tested this hypothesis using pancreatic islet-specific ERα knockout (PERαKO) mice and Min6 β-cells in culture with Esr1 knockdown (KD). We found that Esr1-KD promoted reactive oxygen species production that associated with reduced fission/fusion dynamics and impaired mitophagy. Electron microscopy showed mitochondrial enlargement and a pro-fusion phenotype. Mitochondrial cristae and endoplasmic reticulum were dilated in Esr1-KD compared with ERα replete Min6 β-cells. Increased expression of Oma1 and Chop was paralleled by increased oxygen consumption and apoptosis susceptibility in ERα-KD cells. In contrast, ERα overexpression and ligand activation reduced both Chop and Oma1 expression, likely by ERα binding to consensus estrogen-response element sites in the Oma1 and Chop promoters. Together, our findings suggest that ERα promotes β-cell survival and insulin secretion through maintenance of mitochondrial fission/fusion-mitophagy dynamics and EndoRetic function, in part by Oma1 and Chop repression.

Obesity is associated with increased breast cancer (BrCA) incidence. Considering that inactivation of estrogen receptor (ER)α promotes obesity and metabolic dysfunction in women and female mice, understanding the mechanisms and tissue-specific sites of ERα action to combat metabolic-related disease, including BrCA, is of clinical importance. To study the role of ERα in adipose tissue we generated fat-specific ERα knock-out (FERKO) mice. Herein we show that ERα deletion increased adipocyte size, fat pad weight, and tissue expression and circulating levels of the secreted glycoprotein, lipocalin 2 (Lcn2), an adipokine previously associated with BrCA development. Chromatin immunoprecipitation and luciferase reporter studies showed that ERα binds the Lcn2 promoter to repress its expression. Because adipocytes constitute an important cell type of the breast microenvironment, we examined the impact of adipocyte ERα deletion on cancer cell behavior. Conditioned medium from ERα-null adipocytes and medium containing pure Lcn2 increased proliferation and migration of a subset of BrCA cells in culture. The proliferative and promigratory effects of ERα-deficient adipocyte-conditioned medium on BrCA cells was reversed by Lcn2 deletion. BrCA cell responsiveness to exogenous Lcn2 was heightened in cell types where endogenous Lcn2 expression was minimal, but components of the Lcn2 signaling pathway were enriched, i.e. SLC22A17 and 3-hydroxybutyrate dehydrogenase (BDH2). In breast tumor biopsies from women diagnosed with BrCA we found that BDH2 expression was positively associated with adiposity and circulating Lcn2 levels. Collectively these data suggest that reduction of ERα expression in adipose tissue promotes adiposity and is linked with the progression and severity of BrCA via increased adipocyte-specific Lcn2 production and enhanced tumor cell Lcn2 sensitivity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Estrogen enables uterine proliferation, which depends on synthesis of the IGF1 growth factor. This proliferation and IGF1 synthesis requires the estrogen receptor (ER), which binds directly to target DNA sequences (estrogen-responsive elements or EREs), or interacts with other transcription factors, such as AP1, to impact transcription. We observe neither uterine growth nor an increase in Igf1 transcript in a mouse with a DNA-binding mutated ER alpha (KIKO), indicating that both Igf1 regulation and uterine proliferation require the DNA binding function of the ER. We identified several potential EREs in the Igf1 gene, and chromatin immunoprecipitation analysis revealed ER alpha binding to these EREs in wild type but not KIKO chromatin. STAT5 is also reported to regulate Igf1; uterine Stat5a transcript is increased by estradiol (E(2)), but not in KIKO or alpha ERKO uteri, indicating ER alpha- and ERE-dependent regulation. ER alpha binds to a potential Stat5a ERE. We hypothesize that E(2) increases Stat5a transcript through ERE binding; that ER alpha, either alone or together with STAT5, then acts to increase Igf1 transcription; and that the resulting lack of IGF1 impairs KIKO uterine growth. Treatment with exogenous IGF1, alone or in combination with E(2), induces proliferation in wild type but not KIKO uteri, indicating that IGF1 replacement does not rescue the KIKO proliferative response. Together, these observations suggest in contrast to previous in vitro studies of IGF-1 regulation involving AP1 motifs that direct ER alpha-DNA interaction is required to increase Igf1 transcription. Additionally, full ER alpha function is needed to mediate other cellular signals of the growth factor for uterine growth.

Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ERalpha, a mutant ERalpha (E207A/G208A) that selectively lacks ERE binding, or ERalpha null. In the compound heterozygote (AA/-) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17beta-estradiol (E2). The uterine epithelial proliferative response to E2 and 4-hydroxytamoxifen was retained in the AA/-females, and uterine luminal epithelial height increased commensurate with the extent of ERalpha signaling. This proliferative response was confirmed by 5-bromo-2'-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathway-responsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ERalpha (E207A/G208A) mutant. These results indicate that nonclassical ERalpha signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ERalpha plays a critical physiologic role in the uterine response to estrogen.

In the uterus insulin-like growth factor-1 (IGF-1) signaling can be initiated by estradiol acting through its nuclear receptor (estrogen receptor (ER)) to stimulate the local synthesis of IGF-1. Conversely, in vitro studies have demonstrated that estradiol-independent ER transcriptional activity can be induced by IGF-1 signaling, providing evidence for a cross-talk mechanism between IGF-1 and ER. To investigate whether ER alpha is required for uterine responses to IGF-1 in vivo, both wild-type (WT) and ER alpha knockout (alpha ERKO) mice were administered IGF-1, and various uterine responses to IGF-1 were compared. In both WT and alpha ERKO mice, IGF-1 treatment resulted in phosphorylation of uterine IGF-1 receptor (IGF-1R) and formation of an IGF-1R/insulin receptor substrate-1/ phosphatidylinositol 3-kinase signaling complex. In addition, IGF-1 stimulated phosphorylation of uterine Akt and MAPK in both WT and alpha ERKO mice. However, IGF-1 treatment stimulated BrdUrd incorporation and proliferating cell nuclear antigen expression in WT uteri only. To determine whether ER alpha can be activated in vivo by IGF-1 signaling, transgenic mice carrying a luciferase gene driven by two estrogen response elements (ERE-luciferase mice) were utilized. Treatment of ovariectomized ERE-luciferase mice with IGF-1 resulted in an increase in uterine luciferase activity that was attenuated in the presence of the ER antagonist ICI 182,780. Together these data demonstrate that 1) functional signaling proximal to IGF-1R is maintained in the alpha ERKO mouse uterus, 2) ER alpha is necessary for IGF-1 induction of uterine nuclear proliferative responses, and 3) cross-talk between IGF-1R and ER signaling pathways exists in vivo.