Multiple tissue-specific epigenetic alterations regulate persistent gene expression changes following developmental DES exposure in mouse reproductive tissues.

Clinically, developmental exposure to the endocrine disrupting chemical, diethylstilboestrol (DES), results in long-term male and female infertility. Experimentally, developmental exposure to DES results in abnormal reproductive tract phenotypes in male and female mice. Previously, we reported that neonatal DES exposure causes ERα-mediated aberrations in the transcriptome and in DNA methylation in seminal vesicles (SVs) of adult mice. However, only a subset of DES-altered genes could be explained by changes in DNA methylation. We hypothesized that alterations in histone modification may also contribute to the altered transcriptome during SV development. To test this idea, we performed a series of genome-wide analyses of mouse SVs at pubertal and adult developmental stages in control and DES-exposed wild-type and ERα knockout mice. Neonatal DES exposure altered ERα-mediated mRNA and lncRNA expression in adult SV, including genes encoding chromatin-modifying proteins that can impact histone H3K27ac modification. H3K27ac patterns, particularly at enhancers, and DNA methylation were reprogrammed over time during normal SV development and after DES exposure. Some of these reprogramming changes were ERα-dependent, but others were ERα-independent. A substantial number of DES-altered genes had differential H3K27ac peaks at nearby enhancers. Comparison of gene expression changes, H3K27ac marks and DNA methylation marks between adult SV and adult uterine tissue from ovariectomized mice neonatally exposed to DES revealed that most of the epigenetic changes and altered genes were distinct in the two tissues. These findings indicate that the effects of developmental DES exposure cause reprogramming of reproductive tract tissue differentiation through multiple epigenetic mechanisms.

A Novel Mouse Model to Analyze Non-Genomic ERα Physiological Actions.

Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

The genomic regulatory elements for estrogen receptor alpha transactivation-function-1 regulated genes.

Estrogen receptor alpha (ERα) is a ligand-dependent transcription regulator, containing two transactivation functional domains, AF-1 and AF-2. The selective estrogen receptor modulators (SERMs), including 4-hydroxytamoxifen (4OHT), activate AF-1 preferentially rather than AF-2. However, it is unclear whether this specific function is related to the tissue-selective functionality of SERMs. Moreover, there is no information determining AF-1-dependent estrogenic-genes existing in tissues. We sought to identify AF-1-dependent estrogenic-genes using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an AF-2 disrupted estradiol (E2)-insensitive mutant ERα, but AF-1-dependent transcription can be activated by the estrogen-antagonists, fulvestrant (ICI) and 4OHT. Gene profiling and ChIP-Seq analysis identified Klk1b21 as an ICI-inducible gene in AF2ERKI uterus. The regulatory activity was analyzed further using a cell-based reporter assay. The 5'-flanking 0.4k bp region of Klk1b21 gene responded as an ERα AF-1-dependent estrogen-responsive promoter. The 150 bp minimum ERα binding element (EBE) consists of three direct repeats. These three half-site sequences were essential for the ERα-dependent transactivation and were differentially recognized by E2 and 4OHT for the gene activation. This response was impaired when the minimum EBE was fused with a thymidine-kinase promoter but could be restored by fusion with the 100 bp minimum transcription initiation element (TIE) of Klk1b21, suggesting that the cooperative function of EBE and TIE is essential for mediating AF-1-dependent transactivation. These findings provide the first in vivo evidence that endogenous ERα AF-1 dominant estrogenic-genes exist in estrogen-responsive organs. Such findings will aid in understanding the mechanism of ERα-dependent tissue-selective activity of SERMs.

ESR1 Mutations Associated With Estrogen Insensitivity Syndrome Change Conformation of Ligand-Receptor Complex and Altered Transcriptome Profile.

Estrogen insensitivity syndrome (EIS) arises from rare mutations in estrogen receptor-α (ERα, encoded by ESR1 gene) resulting in the inability of estrogen to exert its biological effects. Due to its rarity, mutations in ESR1 gene and the underlying molecular mechanisms of EIS have not been thoroughly studied. Here, we investigate known ESR1 mutants, Q375H and R394H, associated with EIS patients using in vitro and in vivo systems. Comparison of the transcriptome and deoxyribonucleic acid methylome from stable cell lines of both Q375H and R394H clinical mutants shows a differential profile compared with wild-type ERα, resulting in loss of estrogen responsiveness. Molecular dynamic simulation shows that both ESR1 mutations change the ERα conformation of the ligand-receptor complexes. Furthermore, we generated a mouse model Esr1-Q harboring the human mutation using CRISPR/Cas9 genome editing. Female and male Esr1-Q mice are infertile and have similar phenotypes to αERKO mice. Overall phenotypes of the Esr1-Q mice correspond to those observed in the patient with Q375H. Finally, we explore the effects of a synthetic progestogen and a gonadotropin-releasing hormone inhibitor in the Esr1-Q mice for potentially reversing the impaired female reproductive tract function. These findings provide an important basis for understanding the molecular mechanistic consequences associated with EIS.

A distal super enhancer mediates estrogen-dependent mouse uterine-specific gene transcription of Igf1 (insulin-like growth factor 1).

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.

Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Polycystic ovary syndrome (PCOS) is a hypothalamic-pituitary-gonadal (HPG) axis disorder. PCOS symptoms most likely result from a disturbance in the complex feedback regulation system of the HPG axis, which involves gonadotrophic hormones and ovarian steroid hormones. However, the nature of this complex and interconnecting feedback regulation makes it difficult to dissect the molecular mechanisms responsible for PCOS phenotypes. Global estrogen receptor α (ERα) knockout (KO) mice exhibit a disruption of the HPG axis, resulting in hormonal dysregulation in which female ERα KO mice have elevated levels of serum estradiol (E2), testosterone, and LH. The ERα KO females are anovulatory and develop cystic hemorrhagic ovaries that are thought to be due to persistently high circulating levels of LH from the pituitary. However, the role of ERα in the pituitary is still controversial because of the varied phenotypes reported in pituitary-specific ERα KO mouse models. Therefore, we developed a mouse model where ERα is reintroduced to be exclusively expressed in the pituitary on the background of a global ERα-null (PitERtgKO) mouse. Serum E2 and LH levels were normalized in PitERtgKO females and were comparable to wild-type serum levels. However, the ovaries of PitERtgKO adult mice displayed a more overt cystic and hemorrhagic phenotype when compared with ERα KO littermates. We determined that anomalous sporadic LH secretion caused the severe ovarian phenotype of PitERtgKO females. Our observations suggest that pituitary ERα is involved in the estrogen negative feedback regulation, whereas hypothalamic ERα is necessary for the precise control of LH secretion. Uncontrolled, irregular LH secretion may be the root cause of the cystic ovarian phenotype with similarities to PCOS.-Arao, Y., Hamilton, K. J., Wu, S.-P., Tsai, M.-J., DeMayo, F. J., Korach, K. S. Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Negative elongation factor is essential for endometrial function.

Pausing of RNA polymerase II (Pol II) during early transcription, mediated by the negative elongation factor (NELF) complex, allows cells to coordinate and appropriately respond to signals by modulating the rate of transcriptional pause release. Promoter proximal enrichment of Pol II occurs at uterine genes relevant to reproductive biology; thus, we hypothesized that pausing might impact endometrial response by coordinating hormonal signals involved in establishing and maintaining pregnancy. We deleted the NELF-B subunit in the mouse uterus using PgrCre (NELF-B UtcKO). Resulting females were infertile. Uterine response to the initial decidual stimulus of NELF-B UtcKO was similar to that of control mice; however, subsequent full decidual response was not observed. Cultured NELF-B UtcKO stromal cells exhibited perturbances in extracellular matrix components and also expressed elevated levels of the decidual prolactin Prl8a2, as well as altered levels of transcripts encoding enzymes involved in prostaglandin synthesis and metabolism. Because endometrial stromal cell decidualization is also critical to human reproductive health and fertility, we used small interfering to suppress NELF-B or NELF-E subunits in cultured human endometrial stromal cells, which inhibited decidualization, as reflected by the impaired induction of decidual markers PRL and IGFBP1. Overall, our study indicates NELF-mediated pausing is essential to coordinate endometrial responses and that disruption impairs uterine decidual development during pregnancy.-Hewitt, S. C., Li, R., Adams, N., Winuthayanon, W., Hamilton, K. J., Donoghue, L. J., Lierz, S. L., Garcia, M., Lydon, J. P., DeMayo, F. J., Adelman, K., Korach, K. S. Negative elongation factor is essential for endometrial function.

N-terminal transactivation function, AF-1, of estrogen receptor alpha controls obesity through enhancement of energy expenditure.

OBJECTIVE: Studies using the estrogen receptor alpha (ERα) knock-out (αERKO) mice have demonstrated that ERα plays a crucial role in various estrogen-mediated metabolic regulations. ERα is a ligand dependent transcription regulator and its activity is regulated by estrogenic compounds. ERα consists of two transcriptional activation domains, AF-1 and AF-2. The activities of these domains are regulated through different mechanisms; however, the specific physiological role in metabolic regulation by these domains is still unclear. METHODS: We utilized an ERα AF-2 mutant knock-in mouse (AF2ERKI) to evaluate the physiological functionality of ERα transactivation domains. Due to the estrogen insensitive AF-2 mutation, the phenotypes of AF2ERKI mice are seemingly identical to the global αERKO including obesity in the females. Distinct from the αERKO, the AF-1 function of AF2ERKI mice can be activated by tamoxifen (Tam). Ovariectomized (OVX) AF2ERKI and WT females were treated with Tam and fed a high-fat diet (HFD) for 10 weeks. Additionally, indirect calorimetric analysis was performed using metabolic chambers with food intake and locomotor activity recorded for Tam-treated AF2ERKI and αERKO females. RESULTS: Obesity in HFD-fed AF2ERKI females was prevented by Tam treatment; particularly, inguinal fat accumulation was strongly blocked by Tam treatment. Alterations in fat metabolism genes, however, were not found in either inguinal fat nor visceral fat to be Tam-regulated, even though fat accumulation was strongly reduced by Tam treatment. Indirect calorimetric analysis revealed that without alteration of food intake and locomotor activity Tam treatment increased energy expenditure in AF2ERKI but not αERKO females. CONCLUSIONS: These results suggest that the activation of ERα AF-1 prevents fat accumulation. The prevention of obesity through AF-1 is mediated by induction of energy expenditure rather than ERα AF-1 functionality of lipid metabolism gene regulation in fat tissues.

DNA methylation and transcriptome aberrations mediated by ERα in mouse seminal vesicles following developmental DES exposure.

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERα) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERα mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in αERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health.

Early Endometriosis in Females Is Directed by Immune-Mediated Estrogen Receptor α and IL-6 Cross-Talk.

Endometriosis is a gynecological disease that negatively affects the health of 1 in 10 women. Although more information is known about late stage disease, the early initiation of endometriosis and lesion development is poorly understood. Herein, we use a uterine tissue transfer mouse model of endometriosis to examine early disease development and its dependence on estradiol (E2) and estrogen receptor (ER) α within 72 hours of disease initiation. Using wild-type and ERα knockout mice as hosts or donors, we find substantial infiltration of neutrophils and macrophages into the peritoneal cavity. Examining cell infiltration, lesion gene expression, and peritoneal fluid, we find that E2/ERα plays a minor role in early lesion development. Immune-mediated signaling predominates E2-mediated signaling, but 48 hours after the initiation of disease, a blunted interleukin (IL)-6-mediated response is found in developing lesions lacking ERα. Our data provide evidence that the early initiation of endometriosis is predominantly dependent on the immune system, whereas E2/ERα/IL-6-mediated cross-talk plays a partial role. These findings suggest there are two phases of endometriosis-an immune-dependent phase and a hormone-dependent phase, and that targeting the innate immune system could prevent lesion attachment in this susceptible population of women.

Experimental models for evaluating non-genomic estrogen signaling.

Non-genomic effects of estrogen receptor α (ERα) signaling have been described for decades. However, the mechanisms and physiological processes resulting solely from non-genomic signaling are poorly understood. Challenges in studying these effects arise from the strongly nucleophilic tendencies of estrogen receptor, and many approaches to excluding ERα from the nucleus have been explored over the years. In this review, we discuss past strategies for studying ERα's non-genomic action and current models, specifically H2NES ERα, first described by Burns et al. (2011). In vitro and preliminary in vivo data from H2NES ERα and H2NES mice suggest a promising avenue for pinpointing specific non-genomic ERα action.

Role of ERα in Mediating Female Uterine Transcriptional Responses to IGF1.

Estrogen (E2) signaling through its nuclear receptor, E2 receptor α (ERα) increases insulinlike growth factor 1 (IGF1) in the rodent uterus, which then initiates further signals via the IGF1 receptor. Directly administering IGF1 results in similar biological and transcriptional uterine responses. Our studies using global ERα-null mice demonstrated a loss of uterine biological responses of the uterus to E2 or IGF1 treatment, while maintaining transcriptional responses to IGF1. To address this discrepancy in the need for uterine ERα in mediating the IGF1 transcriptional vs growth responses, we assessed the IGF1 transcriptional responses in PgrCre+Esr1f/f (called ERαUtcKO) mice, which selectively lack ERα in progesterone receptor (PGR) expressing cells, including all uterine cells, while maintaining ERα expression in other tissues and cells that do not express Pgr. Additionally, we profiled IGF1-induced ERα binding sites in uterine chromatin using chromatin immunoprecipitation sequencing. Herein, we explore the transcriptional and molecular signaling that underlies our findings to refine our understanding of uterine IGF1 signaling and identify ERα-mediated and ERα-independent uterine transcriptional responses. Defining these mechanisms in vivo in whole tissue and animal contexts provides details of nuclear receptor mediated mechanisms that impact biological systems and have potential applicability to reproductive processes of humans, livestock and wildlife.

Estrogen Hormone Biology.

The hormone estrogen is involved in both female and male reproduction, as well as numerous other biological systems including the neuroendocrine, vascular, skeletal, and immune systems. Therefore, it is also implicated in many different diseases and conditions such as infertility, obesity, osteoporosis, endometriosis, and a variety of cancers. Estrogen works through its two distinct nuclear receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß). Various transcriptional regulation mechanisms have been identified as the mode of action for estrogen, mainly the classical mechanism with direct DNA binding but also a nongenomic mode of action and one using tethered or indirect binding. The expression profiles of ERα and ERß are unique with the primary sites of ERα expression being the uterus and pituitary gland and the main site of ERß expression being the granulosa cells of the ovary. Mouse models with knockout or mutation of Esr1 and Esr2 have furthered our understanding of the role of each individual receptor plays in physiology. From these studies, it is known that the primary roles for ERα are in the uterus and neuroendocrine system, as female mice lacking ERα are infertile due to impaired ovarian and uterine function, whereas female mice lacking ERß are subfertile due to ovarian defects. The development of effective therapies for estrogen-related diseases has relied on an understanding of the physiological roles and mechanistic functionalities of ERα and ERß in human health and disease.

SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24+ CD49fhi Mammary Stem Cell-Enriched Compartment.

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

Estrogen hormone physiology: reproductive findings from estrogen receptor mutant mice.

Estrogen receptors (ERs) play a crucial role in reproduction and normal physiology. The two sub-types of ER (ERα and ß) are expressed in various levels in different tissues and selective cell types. Gene targeting technology allowed us to produce lines of mice with disrupted ERα (αERKO) and ERß genes (ßERKO) as well as a compound αßERKO in the whole body. Male and female αERKO mice are infertile. Estrogen, EGF and IGF-1 treatments failed to induce uterine growth and DNA synthesis in αERKO uteri. αERKO females are infertile due to hypoplastic uteri and hyperemic ovaries with no corpora lutea due to persistent LH stimulation from loss of negative feedback. αERKO males are infertile, with testicular atrophy and seminiferous tubule dysmorphogenesis producing decreased spermatogenesis and inactive sperm. ßERKO females show arrested folliculogenesis and subfertility. Ovarian analyses indicate differential gene expression related to ovulatory stimulation deficits including lack of LH, PR, Cyp19 and Cox2 expression. A unique ovarian phenotype is found only in αßERKO females showing transdifferentiation of granulosa cells to Sertoli cells. We describe here several novel mouse models which possess ERα gene modification. To understand ERα function in uterine endometrial epithelial cells, we generated a tissue selective ERα gene disrupted mouse model, the uterine epithelial-specific ERα knockout (UtEpiαERKO). To understand the physiological role of ERα functional domains, we generated a mouse model with a mutation in the ligand dependent transcription activation domain of ERα (AF2ERKI). Findings from the ERα mutant mice suggest that the absence of functional ERα is not lethal and results in significant endocrine effects and altered physiological processes.

Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle.

BACKGROUND: Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes. OBJECTIVES: We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression. METHODS: We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice. RESULTS: The DNA methylation status at four specific CpGs (-160, -237, -306, and -367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (-449 and -459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers-DNMT3A, MBD2, and HDAC2-increased in the SV of DES-exposed WT mice. CONCLUSION: DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV. CITATION: Li Y, Hamilton KJ, Lai AY, Burns KA, Li L, Wade PA, Korach KS. 2014. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle. Environ Health Perspect 122:262-268; http://dx.doi.org/10.1289/ehp.1307351.

A single gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts the adult uterine response to estradiol in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) given as a cotreatment with estrogen exhibits antiestrogenic properties on the rodent adult uterus, but less is understood regarding hormonal responsiveness of the adult uterus from animals having been exposed to TCDD during critical periods of development. We characterized the inhibitory effects of TCDD (T) exposure at gestational day 15 (GD15), 4 weeks, and 9 weeks of age (TTT) on the adult uterus following hormone treatment. TTT-exposed mice in response to hormone treatment exhibited a blunted weight increase, had fewer uterine glands, displayed morphological anomalies, and had marked decreases in the hormonal regulation of genes involved in fluid transport (Aqp3 and Aqp5), cytoarchitectural (Dsc2 and Sprr2A), and immune (Lcn2 and Ltf) regulation. To determine if the 9-week exposure was responsible for the blunted uterine response, due to the 7- to 11-day half-life of TCDD in mice, a second set of experiments was performed to examine exposure to TCDD given at GD15, GD15 only (cross-fostered at birth), only during lactation (cross-fostered at birth), or at GD15 and 4 weeks of age. Our studies demonstrate that a single developmental TCDD exposure at GD15 is sufficient to elicit a blunted adult uterine response to estradiol and is due in part to fewer gland numbers and the reduced expression of forkhead box A2 (FoxA2), a gene involved in gland development. Together, these results provide insight regarding the critical nature of in utero exposure and the potential impact on ensuing uterine biology and reproductive health later in life.

Estrogen receptor α L543A,L544A mutation changes antagonists to agonists, correlating with the ligand binding domain dimerization associated with DNA binding activity.

A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region, and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2, and the configuration of H12 is ligand-inducible to an active or inactive form. We demonstrated previously that the ERα mutant (AF2ER) possessing L543A,L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However, it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI-dependent estrogen-responsive element-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI-dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI-dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI-dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT, and raloxifene were parallel with the degree of AF2ER-LBD homodimerization, supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of H12-mutated ERα.

The absence of ER-ß results in altered gene expression in ovarian granulosa cells isolated from in vivo preovulatory follicles.

Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER)-ß is highly expressed in ovarian granulosa cells, and mice lacking ER-ß are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and, although informative, provides confounding results due to the heterogeneous cell types present including granulosa and theca cells and oocytes and exposure to in vitro conditions. Herein we isolated preovulatory granulosa cells from wild-type (WT) and ERß-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of pregnant mare serum gonadotropin (mimicking FSH) and pregnant mare serum gonadotropin/human chorionic gonadotropin (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERß-null ovaries. ERß-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERß in granulosa cells. LH-responsive genes including Abcb1b and Fam110c show reduced expression in ERß-null granulosa cells; however, novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggest that granulosa cells from ERß-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation.