ERß Regulation of Indian Hedgehog Expression in the First Wave of Ovarian Follicles.

Increased activation of ovarian primordial follicles in Erß knockout (ErßKO) rats becomes evident as early as postnatal day 8.5. To identify the ERß-regulated genes that may control ovarian primordial follicle activation, we analyzed the transcriptome profiles of ErßKO rat ovaries collected on postnatal days 4.5, 6.5, and 8.5. Compared to wildtype ovaries, ErßKO ovaries displayed dramatic downregulation of Indian hedgehog (Ihh) expression. IHH-regulated genes, including Hhip, Gli1, and Ptch1, were also downregulated in ErßKO ovaries. This was associated with a downregulation of steroidogenic enzymes Cyp11a1, Cyp19a1, and Hsd17b1. The expression of Ihh remained very low in ErßKO ovaries despite the high levels of Gdf9 and Bmp15, which are known upregulators of Ihh expression in the granulosa cells of activated ovarian follicles. Strikingly, the downregulation of the Ihh gene in ErßKO ovaries began to disappear on postnatal day 16.5 and recovered on postnatal day 21.5. In rat ovaries, the first wave of primordial follicles is rapidly activated after their formation, whereas the second wave of primordial follicles remains dormant in the ovarian cortex and slowly starts activating after postnatal day 12.5. We localized the expression of Ihh mRNA in postnatal day 8.5 wildtype rat ovaries but not in the age-matched ErßKO ovaries. In postnatal day 21.5 ErßKO rat ovaries, we detected Ihh mRNA mainly in the activated follicles in the ovaries' peripheral regions. Our findings indicate that the expression of Ihh in the granulosa cells of the activated first wave of ovarian follicles depends on ERß.

DOT1L Methyltransferase Regulates Calcium Influx in Erythroid Progenitor Cells in Response to Erythropoietin.

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca2+) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1lKO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1lKO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca2+ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca2+ influx by TRPC2, Dot1lKO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca2+ influx. Such heightened Ca2+ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.

DOT1L Mediated Gene Repression in Extensively Self-Renewing Erythroblasts.

DOT1L is essential for embryonic hematopoiesis but the precise mechanisms of its action remain unclear. The only recognized function of DOT1L is histone H3 lysine 79 (H3K79) methylation, which has been implicated in both transcriptional activation and repression. We observed that deletion of the mouse Dot1L gene (Dot1L-KO) or selective mutation of its methyltransferase domain (Dot1L-MM) can differentially affect early embryonic erythropoiesis. However, both mutations result in embryonic lethality by mid-gestation and growth of hematopoietic progenitor cells (HPCs) is similarly affected in extensively self-renewing erythroblast (ESRE) cultures established from yolk sac cells. To understand DOT1L-mediated gene regulation and to clarify the role of H3K79 methylation, we analyzed whole transcriptomes of wildtype and Dot1L-mutant ESRE cells. We observed that more than 80% of the differentially expressed genes (DEGs) were upregulated in the mutant ESRE cells either lacking the DOT1L protein or the DOT1L methyltransferase activity. However, approximately 45% of the DEGs were unique to either mutant group, indicating that DOT1L possesses both methyltransferase-dependent and -independent gene regulatory functions. Analyses of Gene Ontology and signaling pathways for the DEGs were consistent, with DEGs that were found to be common or unique to either mutant group. Genes related to proliferation of HPCs were primarily impacted in Dot1L-KO cells, while genes related to HPC development were affected in the Dot1L-MM cells. A subset of genes related to differentiation of HPCs were affected in both mutant groups of ESREs. Our findings suggest that DOT1L primarily acts to repress gene expression in HPCs, and this function can be independent of its methyltransferase activity.

Transcriptional regulation of Satb1 in mouse trophoblast stem cells.

SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages that emerge during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain the trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated an active state of the promoter. A novel cis-regulatory region with active histone marks was identified ∼21 kbp upstream of the variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromosome conformation capture analysis confirmed looping of this distant regulatory region into the proximal promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close proximity to SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.

Granulosa cell genes that regulate ovarian follicle development beyond the antral stage: The role of estrogen receptor ß.

Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erßnull ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERß is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erßnull mutant model that fails to show follicle maturation was also included in order to identify the ERß-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erßnull GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erßnull GCs. These latter ERß-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERß-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erßnull GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERß.

Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity.

DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

Disruption of ESR1 alters the expression of genes regulating hepatic lipid and carbohydrate metabolism in male rats.

The liver helps maintain energy homeostasis by synthesizing and storing glucose and lipids. Gonadal steroids, particularly estrogens, play an important role in regulating metabolism. As estrogens are considered female hormones, metabolic disorders related to the disruption of estrogen signaling have mostly been studied in females. Estrogen receptor alpha (ESR1) is the predominant receptor in both the male and female liver, and it mediates the hepatic response to estrogens. Loss of ESR1 increases weight gain and obesity in female rats, while reducing the normal growth in males. Although Esr1-/- male rats have a reduced body weight, they exhibit increased adipose deposition and impaired glucose tolerance. We further investigated whether these metabolic disorders in Esr1-/- male rats were linked with the loss of transcriptional regulation by ESR1 in the liver. To identify the ESR-regulated genes, RNA-sequencing was performed on liver mRNAs from wildtype and Esr1-/- male rats. Based on an absolute fold change of ≥2 with a p-value ≤ 0.05, a total of 706 differentially expressed genes were identified in the Esr1-/- male liver: 478 downregulated, and 228 upregulated. Pathway analyses demonstrate that the differentially expressed genes include transcriptional regulators (Cry1, Nr1d1, Nr0b2), transporters (Slc1a2), and regulators of biosynthesis (Cyp7b1, Cyp8b1), and hormone metabolism (Hsd17b2, Sult1e1). Many of these genes are also integral parts of the lipid and carbohydrate metabolism pathways in the liver. Interestingly, certain critical regulators of the metabolic pathways displayed a sexual dimorphism in expression, which may explain the divergent weight gain in Esr1-/- male and female rats despite common metabolic dysfunctions.

Liver transcriptome data of Esr1 knockout male rats reveals altered expression of genes involved in carbohydrate and lipid metabolism.

Estrogens are traditionally considered to be female sex steroid hormones and most of the studies examining estrogen regulation of metabolic function in the liver have been conducted in females. However, the liver expresses high levels of estrogen receptor alpha (ESR1) in both males and females, which mediates the hepatic response to estrogens. In this data article, we investigated whether metabolic disorders in Esr1 knockout (Esr1-/-) male rats were linked with loss of transcriptional regulation by ESR1 in liver. To identify the ESR1 regulated genes in the mutant liver, RNA-sequencing was performed on liver RNAs purified from young male rats. The raw data were analyzed using the CLC Genomics Workbench and high-quality RNA-sequencing reads were aligned to the Rattus norvegicus genome. Transcriptome data obtained from Esr1-/- liver RNAs were compared to that of wild type rats. Based on an absolute fold change of 2 with a p-value ≤ 0.05, a total of 618 differentially expressed genes were identified in the Esr1-/- male liver. Pathway analyses demonstrated that the majority of differentially expressed genes are regulators of carbohydrate and lipid metabolism in the liver. These differentially expressed genes and their potential roles were further examined in a companion manuscript, "Disruption of ESR1 alters the expression of genes regulating hepatic lipid and carbohydrate metabolism in male rats" (Khristi et al., 2018).

Differentially regulated genes in Esr2-mutant rat granulosa cells.

RNA seq analyses were performed in granulosa cells (GCs) collected from gonadotropin treated ESR2 mutant rats. Data obtained from a null mutant with Esr2 exon 3 deletion (∆3) and another DNA binding domain (DBD) mutant with exon 4 deletion (∆4) were compared to that of wildtype (WT) rats. The raw data were analyzed using CLC genomics workbench. High quality RNA-sequencing reads were aligned to the Rattus norvegicus genome. Differentially expressed genes in ∆3 or ∆4 Esr2-mutant GCs were identified based on the following criteria: FDR p-Value ≤0.05 and an absolute fold change of 2. Fewer differentially expressed genes were identified in ∆3 compared to the ∆4 mutant group. As both mutant groups demonstrated a common phenotype of ovulation failure, differentially expressed genes common to both in ∆3 and ∆4 mutant rats were emphasized and further analyzed in the companion article "ESR2 regulates granulosa cell genes essential for follicle maturation and ovulation" [1].

ESR2 regulates granulosa cell genes essential for follicle maturation and ovulation.

Estrogen receptor 2 (ESR2) plays a critical role in folliculogenesis and ovulation. Disruption of ESR2-function in the rats results in female infertility due to failure of ovulation. Ovulation failure occurred in two distinct rat models, a null mutant and a DNA binding domain (DBD) mutant of ESR2, indicating that transcriptional regulation by ESR2 is indispensable for ovulation. To define the regulatory role of ESR2 in preovulatory follicular maturation and ovulation, we investigated ovarian responsiveness to exogenous gonadotropins in prepubertal females. Granulosa cells (GCs) play a vital role in follicle maturation and ovulation, and ESR2-dependent estrogen signaling is predominant in GCs, therefore, we examined the differential expression of gonadotropin-induced genes in GCs. Of 32,623 genes detected by RNA-sequencing, 1696 were differentially expressed in Esr2-mutant rats (789 downregulated, and 907 upregulated, absolute fold change 2, FDR p < 0.05). Molecular pathway analyses indicated that these differentially expressed genes are involved in steroidogenesis, follicle maturation, and ovulation. Many of these genes are known regulators of ovarian function and a subset were also disrupted in Esr2-mutant mice. Interestingly, Kiss1 was identified as one of the differentially expressed genes implicating a potential role within the follicle and its regulation by ESR2. Our findings indicate that ESR2 regulates key genes in GCs that are essential for follicle maturation and ovulation in the rat.

Defining the Role of Estrogen Receptor ß in the Regulation of Female Fertility.

Estrogens are essential hormones for the regulation of fertility. Cellular responses to estrogens are mediated by estrogen receptor α (ESR1) and estrogen receptor ß (ESR2). In mouse and rat models, disruption of Esr1 causes infertility in both males and females. However, the role of ESR2 in reproductive function remains undecided because of a wide variation in phenotypic observations among Esr2-mutant mouse strains. Regulatory pathways independent of ESR2 binding to its cognate DNA response element have also been implicated in ESR2 signaling. To clarify the regulatory roles of ESR2, we generated two mutant rat models: one with a null mutation (exon 3 deletion, Esr2ΔE3) and the other with an inframe deletion selectively disrupting the DNA binding domain (exon 4 deletion, Esr2ΔE4). In both models, we observed that ESR2-mutant males were fertile. ESR2-mutant females exhibited regular estrous cycles and could be inseminated by wild-type (WT) males but did not become pregnant or pseudopregnant. Esr2-mutant ovaries were small and differed from WT ovaries by their absence of corpora lutea, despite the presence of follicles at various stages of development. Esr2ΔE3- and Esr2ΔE4-mutant females exhibited attenuated preovulatory gonadotropin surges and did not ovulate in response to a gonadotropin regimen effective in WT rats. Similarities of reproductive deficits in Esr2ΔE3 and Esr2ΔE4 mutants suggest that DNA binding-dependent transcriptional function of ESR2 is critical for preovulatory follicle maturation and ovulation. Overall, the findings indicate that neuroendocrine and ovarian deficits are linked to infertility observed in Esr2-mutant rats.