United States

Introduction: Estrogen is vital to human reproduction and acts primarily through two receptors: estrogen receptor alpha (ER𝛼, encoded by ESR1) and estrogen receptor beta (ERβ, encoded by ESR2). Surprisingly, very few human ESR1 pathogenic variants have been recognized, despite the existence of &gt;1000 different androgen receptor pathogenic variants causing androgen insensitivity syndrome. Functional analysis of an ESR1 homozygous missense variant (p.Glu375His) in the ER𝛼 ligand-binding domain (LBD) showed a 240-fold decrease in estrogen response in a patient with complete estrogen insensitivity and abnormal pubertal development. We predict that less severe ESR1 variants will present with unexplained infertility. Previously, we performed whole exome sequencing on 200 females with unexplained infertility and identified 4 likely pathogenic ESR1 heterozygous missense variants confirmed by Sanger sequencing, including one variant (p.Thr313Met) in the LBD. In this study, we performed in vitro functional analysis to evaluate if the p.Thr313Met likely pathogenic variant found in a patient with unexplained infertility causes incomplete estrogen insensitivity.
Hypothesis: We hypothesize that the p.Thr313Met likely pathogenic variant in the ERa LBD will result in incomplete estrogen insensitivity.
Methods: The p.Thr313Met variant plasmid was constructed using site-directed mutagenesis (SDM), and confirmed by Sanger sequencing. An in vitro cell model was used to evaluate estrogenic activity. HepG2 cells were transiently transfected with WT ESR1, p.Thr313Met, or p.Glu375His plasmid, along with the Firefly estrogen response element (ERE) luciferase reporter (3X ERE TATA luc) and Renilla luciferase (pRL-TK luc) plasmids using the Effectene transfection protocol. Cells were treated with 17β-estradiol (0.01, 0.1, 1, 10, and 100 nM). A dual luciferase reporter assay was used to compare relative Firefly and Renilla luciferase activity between WT ESR1, p.Thr313Met, and p.Glu375His plasmids using a luminometer. Estrogen dose response curves were generated, and a two-way ANOVA with Tukey&#39;s test was performed to compare the relative luciferase activity across WT and variant plasmids using GraphPad Prism v.9. Differences were considered significant at p&lt;0.05.
Results: Estrogen response curves were generated for WT, p.Thr313Met, and p.Glu375His plasmids. The p.Thr313Met variant showed lower overall activity with a right shifted dose response curve with a statistically significant decrease in relative luciferase activity in comparison to WT (p&lt;0.05). The p.Thr313Met variant was more active at higher doses (1 and 10 nM) with a left shifted dose curve when compared to the p.Glu375His variant (p&lt;0.05).
Conclusion: We have identified a human ESR1 variant that impairs estrogen signaling in vitro, suggesting incomplete estrogen insensitivity as a putative mechanism for unexplained infertility. The identification of ESR1 pathogenic variants and the evaluation of their functionality could provide improved diagnosis and genetic counseling for patients with unexplained infertility.

ENDO 2022

ESR1 Pathogenic Variant With Incomplete Estrogen Insensitivity

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Introduction: Estrogen is vital to human reproduction and acts primarily through two receptors: estrogen receptor alpha (ER𝛼, encoded by ESR1) and estrogen receptor beta (ERβ, encoded by ESR2). Surprisingly, very few human ESR1 pathogenic variants have been recognized, despite the existence of >1000 different androgen receptor pathogenic variants causing androgen insensitivity syndrome. Functional analysis of an ESR1 homozygous missense variant (p.Glu375His) in the ER𝛼 ligand-binding domain (LBD) showed a 240-fold decrease in estrogen response in a patient with complete estrogen insensitivity and abnormal pubertal development. We predict that less severe ESR1 variants will present with unexplained infertility. Previously, we performed whole exome sequencing on 200 females with unexplained infertility and identified 4 likely pathogenic ESR1 heterozygous missense variants confirmed by Sanger sequencing, including one variant (p.Thr313Met) in the LBD. In this study, we performed in vitro functional analysis to evaluate if the p.Thr313Met likely pathogenic variant found in a patient with unexplained infertility causes incomplete estrogen insensitivity.
Hypothesis: We hypothesize that the p.Thr313Met likely pathogenic variant in the ERa LBD will result in incomplete estrogen insensitivity.
Methods: The p.Thr313Met variant plasmid was constructed using site-directed mutagenesis (SDM), and confirmed by Sanger sequencing. An in vitro cell model was used to evaluate estrogenic activity. HepG2 cells were transiently transfected with WT ESR1, p.Thr313Met, or p.Glu375His plasmid, along with the Firefly estrogen response element (ERE) luciferase reporter (3X ERE TATA luc) and Renilla luciferase (pRL-TK luc) plasmids using the Effectene transfection protocol. Cells were treated with 17β-estradiol (0.01, 0.1, 1, 10, and 100 nM). A dual luciferase reporter assay was used to compare relative Firefly and Renilla luciferase activity between WT ESR1, p.Thr313Met, and p.Glu375His plasmids using a luminometer. Estrogen dose response curves were generated, and a two-way ANOVA with Tukey's test was performed to compare the relative luciferase activity across WT and variant plasmids using GraphPad Prism v.9. Differences were considered significant at p<0.05.
Results: Estrogen response curves were generated for WT, p.Thr313Met, and p.Glu375His plasmids. The p.Thr313Met variant showed lower overall activity with a right shifted dose response curve with a statistically significant decrease in relative luciferase activity in comparison to WT (p<0.05). The p.Thr313Met variant was more active at higher doses (1 and 10 nM) with a left shifted dose curve when compared to the p.Glu375His variant (p<0.05).
Conclusion: We have identified a human ESR1 variant that impairs estrogen signaling in vitro, suggesting incomplete estrogen insensitivity as a putative mechanism for unexplained infertility. The identification of ESR1 pathogenic variants and the evaluation of their functionality could provide improved diagnosis and genetic counseling for patients with unexplained infertility.

technical paper

Although obesity is closely associated with reproductive dysfunction, many women with obesity remain eumenorrheic. The degree to which obesity affects menstrual cycle characteristics in this population is limited to endocrine assessments with few data available on how endocrine aberrations align with defects in antral follicle development.We have previously shown alterations in the distribution of recruitable (2-5mm) versus selectable (6-9mm) follicles in those with obesity that were associated with decreased gonadotropin and progesterone production despite normal menstrual cycle length. The objectives of the present study were to further contrast follicular growth and endocrine dynamics between obese and non-obese eumenorrheic women with the goal of determining any alterations in distinct events in folliculogenesis.
21 non-obese (total percent body fat <35%) and 21 obese (total percent body fat ≥35%) reproductive age females with regular menstrual cycles (21-35d) were evaluated by serial ovarian transvaginal ultrasonography and venipuncture every-other-day for one complete inter-ovulatory interval (IOI). The number and diameters of all follicles ≥2mm at each visit were documented offline using the Grid Method. Growth profiles of individual follicles which grew to ≥7mm were assessed using the Identity Method to characterize antral follicle recruitment, selection, and ovulation. Clinical, morphologic, and endocrine features were compared across groups using t-tests and mixed models.
Women with obesity exhibited fewer recruitment events versus non-obese women (1±0.6 vs. 2±0.9; p=0.010). They also had fewer selectable follicles (4±3 vs. 8±6 follicles/participant, p=0.022) which grew faster but remained static longer and regressed slower (all growth rates, p<0.05) compared to their non-obese counterparts. Anti-Mullerian hormone levels were lower in the obese versus non-obese group (4.40±3.01 vs. 5.94±2.49ng/mL, p=0.023). Of the selectable follicles, fewer progressed to ≥10mm in the obese versus non-obese group (1±0.7 vs. 2±0.8 follicles, p=0.030) with ultimately fewer anovulatory follicles achieving dominance (0±0.7 vs. 1±0.9 follicle, p=0.029). Ovulatory follicles in the obese group were selected at smaller diameters (7.5±1.6 vs. 9.5±1.9mm, p=0.001) versus their non-obese counterparts. However, the kinetics of dominant follicle growth were similar between groups. Post-ovulation, progesterone concentrations across the luteal phase were lower in women with obesity (4.95±3.34 vs. 7.85±5.74ng/mL, p=0.001) and the prevalence of luteal phase defects (LPDs) higher versus non-obese women, defined by either integrated (76 vs. 29%, p=0.002) or maximum (71 vs. 24%, p=0.002) progesterone levels.
Eumenorrheic women with obesity display evidence of suppressed follicle dynamics as evidenced by fewer recruitment events, selectable follicles, and anovulatory dominant follicles.  LPDs are more prevalent in women with obesity during natural ovulatory cycles which may underlie subfertility in this population. A smaller size at selection suggests premature responsiveness to luteinizing hormone which may result in suboptimal luteinization. Further studies are needed to elucidate the degree to which these defects impact fertility and fecundity in our increasingly obese demographic.

Women with obesity display defects in follicular recruitment, selection, and dominance alongside luteal phase dysfunction during natural ovulatory cycles

Background: SKYLIGHT 2 (NCT04003142) investigated safety and efficacy of fezolinetant (a neurokinin-3 receptor antagonist) on frequency and severity of moderate-to-severe vasomotor symptoms (VMS) and sleep disturbance. In this analysis, persistence of fezolinetant efficacy was investigated over 52 weeks.
Methods: This double-blind Phase 3 study randomized women aged ≥40–65 years with moderate-to-severe VMS associated with menopause (average of ≥7 hot flashes/day) to once-daily placebo or fezolinetant 30mg or 45mg for 12 weeks. Women completing 12 weeks entered an extension period, with those on placebo re-randomized to fezolinetant 30mg or 45mg (placebo/fezolinetant), and those originally on fezolinetant remaining on their dose for an additional 40-weeks. Fezolinetant efficacy was evaluated vs placebo for 12-weeks through change in VMS frequency, VMS severity, and Patient-reported Outcomes Measurement Information System Sleep Disturbance–Short Form 8b (PROMIS) Total Score. Persistence of efficacy for fezolinetant was evaluated descriptively (without statistical comparisons) over the extension period. Results: The analysis comprised 484 women (fezolinetant 30mg n=166, fezolinetant 45mg n=167, placebo/fezolinetant 30mg n=76, placebo/fezolinetant 45mg n=75). Improvement in VMS frequency and severity observed through week 12 (statistically significant differences vs placebo) was maintained throughout the 52-week total study period for those receiving fezolinetant. Fezolinetant demonstrated further reductions in VMS frequency and severity from baseline to beyond week 12. For VMS frequency, there was a least squares (LS) mean (SE) baseline-to-week 12 reduction of –6.83 (0.39) VMS/day for fezolinetant 30mg and –7.50 (0.39) for 45mg, and a mean (SD) baseline-to-week 52 reduction of –8.03 (4.53) for fezolinetant 30mg and –8.48 (3.98) for 45mg. For VMS severity, LS mean (SE) baseline-to-week 12 reduction was –0.64 (0.06) for 30mg and –0.77 (0.06) for 45mg, and mean (SD) baseline-to-week 52 reduction was –0.83 (0.82) for fezolinetant 30mg, and –0.95 (0.78) for 45mg. Women re-randomized from placebo to fezolinetant experienced a reduction in frequency and severity of VMS consistent with that in women receiving fezolinetant throughout the study. Fezolinetant also reduced PROMIS-assessed sleep disturbance, with a LS mean (SE) baseline-to-week 12 reduction of –4.1 (0.5) for fezolinetant 30mg and –5.5 (0.5) for 45mg (statistically significant difference vs placebo for fezolinetant 45mg) and a mean (SD) baseline-to-week 52 reduction of – 6.3 (7.3) for fezolinetant 30mg and –5.7 (7.9) for 45mg. The safety profile observed for the 40-week extension period was consistent with that of the 12-week placebo-controlled period. Conclusion: Fezolinetant 30mg and 45mg once daily were efficacious for treatment of moderate-to-severe VMS associated with menopause. Efficacy was persistent and reductions in VMS frequency were maintained during the extension period, at levels consistent with weeks 1 through 12. Fezolinetant 45mg improved sleep at week 12 and improvement was maintained through the active treatment extension period. No safety signals of concern were apparent for either fezolinetant dose.

Fezolinetant for Treatment of Moderate-to-severe Vasomotor Symptoms Associated with Menopause: Results from a 52-week Study (Skylight 2)

Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor.  Upon binding to endogenous and exogenous ligands, ERα dimerizes and binds to regulatory regions across the genome known as enhancers.  Enhancers act as nucleation sites for the recruitment of coregulatory proteins and ultimately regulate target gene transcription.  We have previously used global run-on sequencing (GRO-seq) to detect transcription at active enhancers that generates enhancer RNAs (eRNAs).  eRNAs are poorly characterized noncoding RNAs and are thought to regulate enhancer function and gene expression.  To better understand the functions of these eRNAs, we annotated the estrogen-regulated eRNA transcriptome in ERα-positive breast cancer cells using precision nuclear run-on of capped RNA (PRO-cap) and polyA-depleted RNA-seq.  We then cloned selected eRNAs, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9.  eRNAs promoted expression of cognate, but not heterologous, target genes by increasing ERα recruitment and simulating p300-catalyzed H3K27 acetylation at the enhancer.  We identified a ~40 nucleotide functional eRNA regulatory motif (FERM) present in many estrogen-regulated eRNAs that is necessary and sufficient to promote gene expression by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation.  The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers, to exert its effect on enhancer assembly and dynamics.  Overexpression of a targeted eRNA controlling expression of an oncogene increased cell proliferation, implicating eRNAs in regulating breast cancer cell growth.  Together, we provide a genome-wide annotation of estrogen-regulated eRNAs, demonstrate the mechanisms by which targeted eRNAs promote target gene expression in vivo, identify the FERM element within eRNAs that is critical for regulating target gene expression, and show the biological implications of oncogenic eRNAs in breast cancer growth.  Collectively, our results demonstrate the biological and mechanistic roles of eRNAs in breast cancer.
  This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.

Estrogen-regulated Enhancer RNAs Contain a Functional Motif that Controls Enhancer Assembly and Function in Breast Cancer Cells

Invasive lobular carcinoma of the breast (ILC) affects ~40,000 US women annually and is a top ten most diagnosed cancer in women. ~95% of ILC express estrogen receptor alpha (ER), but ILC is associated with poor long-term outcomes and anti-estrogen resistance, suggesting ER function is distinct in ILC cells. We reported that MDC1 (mediator of DNA damage checkpoint 1) is an ILC-specific ER co-regulator required for ER-driven proliferation, and that MDC1 knockdown dysregulates the ER transcriptome in ILC. Mechanisms of MDC1 co-regulator activity are unknown, but must be defined to understand endocrine response and resistance in ILC.
To understand MDC1 co-regulator functions, we profiled ER/MDC1 genomic binding (ie. "cistrome") using CUT&RUN in ER+ ILC cell line MDA MB 134VI (MM134). We identified ~2500 binding sites each for ER and MDC1, but only ~8% of sites were bound by both factors. However, using BETA to integrate cistrome and transcriptome data predicted that both ER and MDC1 activate estrogen-regulated genes with over half of direct ER target genes also direct MDC1 targets (p=9.8x10-375). Among shared ER: MDC1 direct target genes (n=760), 58% had MDC1 binding in the promoter region with ER bound at nearby enhancers, while only 15% were linked to shared ER: MDC1 binding sites, suggesting MDC1 primarily facilitates promoter access for ER-bound enhancers. Further, MDC1 knockdown reduced ER binding at enhancer sites at ER: MDC1 target genes, implicating MDC1 in regulating chromatin access.
Importantly, MDC1 does not have known enzymatic activity to directly regulate chromatin structure. We profiled MDC1-interacting proteins in ILC cells by RIME (Rapid immunoprecipitation mass spectrometry of endogenous proteins), and determined whether knockdown of putative ER: MDC1 partners disrupted ER function. This identified the SWI/SNF complex protein BRG1 (SMARCA4) as a critical ER: MDC1 partner. BRG1 was required for ILC cell growth, and BRG1 knockdown specifically suppressed ER regulation of MDC1-dependent ER target genes.
Taken together, our data suggest that in ILC cells, MDC1 facilitates ER access to and activity at target gene promoters. The BRG1 SWI/SNF complex is a putative ER: MDC1 partner and mediator of MDC1 co-regulator functions. Ongoing research is focused on understanding how MDC1 regulates ILC-specific ER activity by licensing access to ILC-specific target genes, and the role of the SWI/SNF complex in mediating genomic ER: MDC1 activity.

Endocrine Response And Resistance Is Driven By Novel Transcriptional Co-Regulator Activity In Invasive Lobular Carcinoma Of The Breast

During fasting, hepatocytes produce glucose in response to hormonal signals. Glucagon and glucocorticoids are principal hormones secreted during fasting that cooperate in regulating glucose production via increasing hepatic gluconeogenesis. However, how these hormone signals are integrated in hepatocytes and translated to a biological output is unknown. We used genome-wide profiling of gene expression, enhancer dynamics and transcription factor binding in primary mouse hepatocytes to uncover the mode of cooperation between glucagon and corticosterone (the major glucocorticoid in mice). We found that compared to a single treatment with either hormone alone, a dual treatment initiates a pro-gluconeogenic gene program in hepatocytes by synergistically inducing gluconeogenic genes. We show that the mechanism driving synergistic gene expression is based on glucagon-mediated enhancer activation, leading to increased binding of the glucocorticoid receptor (GR) upon corticosterone stimulation. This was shown by glucagon-dependent increases in the active enhancer mark H3K27ac at GR binding sites (both signals were measured genome-wide via chromatin immunoprecipitation sequencing). Thus, glucagon-dependent enhancer activation, mediated by the glucagon-activated transcription factor CREB, assists GR loading to specific enhancers located near synergistic gluconeogenic genes. This 'assisted loading' mechanism is enhancer-specific as GR was able to efficiently bind other enhancers without assistance by CREB. Interestingly, we found that the glucagon-CREB axis does not only activate single enhancers but is also able to activate entire enhancer clusters. Indeed, CREB binding in one enhancer unit within a cluster, led to cluster-wide enhancer activation, thereby assisting the loading of GR in other enhancer units within the cluster that are unbound by CREB directly. These chromatin and gene expression changes collectively lead to synergistic glucose production from hepatocytes in the presence of glucagon and corticosterone. In summary, we show that hepatocytes integrate extracellular signals by an enhancer-specific mechanism: glucagon activates enhancers, thereby assisting the loading of GR upon stimulation by corticosterone. This chromatin-bound integration of endocrine signals leads to synergistic gene induction and a tailored response to fasting.

Hormone-controlled cooperative binding of transcription factors within enhancer clusters drives synergistic induction of fasting-regulated genes

The mineralocorticoid receptor (MR) is a ligand activated transcription factor that plays an important role in cardiovascular physiology and disease. MR transactivation is regulated by ligand-dependent conformational change in the MR and recruitment of coregulator binding partners to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate a candidate MR coregulator, the circadian gene timeless for ligand and cell-selective regulation of MR transactivation. While timeless is not essential for mammalian clock function and does not bind DNA, timeless RNA and protein levels oscillate over 24hr. Co-expression of timeless dose-dependently down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. Timeless also inhibited MR transactivation of synthetic and native gene promoters, and expression of endogenous MR target genes, in H9c2 cardiac myoblasts. Immunofluorescence staining of timeless, and MR showed colocalization to the nucleus in the presence of aldosterone and co-immunoprecipitation which suggested an interaction, although a direct interaction was not confirmed. Taken together, these data suggest timeless is a novel MR coregulatory protein that may contribute to time-of-day-dependent MR transcriptional outputs and suggest new opportunities for the development of MR antagonists with selective actions.

Regulation of mineralocorticoid receptor transactivation by circadian protein timeless.

Background: NAD+-dependent PARP1 (Poly-(ADP-ribosyl)polymerase 1) generates the post-translational modification ADP-Ribosylation (ADPR). Given PARP1-ADPR determines cellular NAD+ availability, which in turn impacts a range of hormonally governed actions, we sought to identify the molecular roles of PARP1 in relation to glucocorticoids in the endocrine sensitive tissue skeletal muscle.
Importance: Activity of the glucocorticoid receptor (GR) is dictated by molecular partners whose actions skew the frequency with which GR produces meaningful transcriptional outcomes. As a result, our molecular understanding of this steroid hormone receptor remains incomplete.
Hypothesis: PARP1 through ADPR actions impacts skeletal muscle phenotype and co-regulates glucocorticoid-mediated transcriptional responses.
Experimental Design: Murine muscle myoblasts (C2C12) or primary murine muscle cultures were treated with PARP inhibitors (PJ34(10uM) or BYK204165(1uM)) or transfected with silencing RNA targeting PARP1. Cells were treated with ± dexamethasone (1uM).
Major Results: Analysis of differentiating muscle revealed PARP1 and ADPR are dynamic at days 0-5 (measured by Western immunoblot. p<0.001; n=6). Chemical inhibition of PARP1 significantly decreased ADPR (Western immunoblot. p<0.001; n=9) but did not prevent myotube formation. To identify if initial ADPR dynamics held consequence for fully differentiated myotubes, cells were treated with a single dose of the PARP1 inhibitor BYK204165 at induction of differentiation. Myotube lysates were collected 5 days later and were subjected to unlabelled SWATH-MS. This measured the abundance of 180 proteins as significantly different in PARP inhibitor-treated cells (n=7). Analyses of these proteins revealed over-representation of pathways governing muscle development (FDR 8.99-08), contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05), and energy metabolism (FDR 2.93-06). Indicating PARP1-ADPR regulates myogenesis and plays a role in the striated muscle contractile response.
ChIP assays for PARP1 binding in cells showed a (30-fold enrichment ±3.2SEM) at GR response elements and detectable ADPR was reduced when exposed to dexamethasone (Western immunoblot n=5). These data support ADPR as responsive to glucocorticoid exposure and PARP1 influences GR recruitment to the genome.
RNAseq of siPARP1 (n=4) versus scrambled myoblasts (n=5) identified differential expression of 165 genes. Gene ontology revealed over-representation in siPARP1 cells of those pathways regulating muscle differentiation, including actin-binding, cytoskeletal structure, and NAD+ binding genes. SiRNA of PARP1 had no influence over the ability of glucocorticoids to activate archetype target genes (DUSP1, MURF1, ATROGIN1 & GILZ), but differentially impacted the expression of 434 other genes.
Conclusions: PARP1 mediated ADPR is critical to the 'early phase' events of muscle cell differentiation. PARP1 influences short-term transcriptional behaviors of the glucocorticoid receptor; in turn, glucocorticoids themselves impact PARP1-genome interaction events. These findings are important for the understanding of general GR mechanisms and have relevance for the widespread functional decline observed during tissue glucocorticoid excess.

PARP1 Contributes to the Glucocorticoid Receptor Transcriptional Response And Influences the Skeletal Muscle Phenotype

The androgen receptor (AR) plays a crucial role in the development and maintenance of the male phenotype, as shown in patients with androgen insensitivity syndrome (AIS). The AR is a nuclear receptor that needs to homodimerize to execute its role as transcription factor. Dimerization can occur through three different modes: via the DNA-binding domain (DBD), via the ligand-binding domain and via an interaction between the LBD and the aminoterminal domain. Dimerization via the DBD is very well known and occurs through the D-box located in the second zinc finger. DBD dimerization is generally accepted for all steroid receptors, but some of them show that D-box mutations do not inactivate them completely. However, for the AR, no monomeric transactivation capacity was described yet. To study the role of D-box independent activity of the AR, we introduced a double point mutation in the D-box that is predicted to disrupt DBD dimerization (ARDmon). These two point mutations were found in separate patients suffering from partial AIS. In vitro studies with this mutant suggest that the remaining activity depends on the androgen response element under investigation. We show that ligand binding was not affected by the D-box mutation. Furthermore, BioID assays indicated no changes in coregulatory interactions. When introducing the corresponding mutation in mice, this led to a phenotype that is intermediate between global ARKO and wild type (WT) males. Based on their anogenital distance (AGD) and the absence of nipples at early age, the ARDmon/Y mice were identified as males. Follow-up of their AGD and body weight, however, revealed clear differences with both WT males and females. Furthermore, hypospadias was observed in the ARDmon/Y mice. Testes were smaller in size compared to the WT males and first analysis showed almost no expression of well-known AR regulated genes. Reminiscent structures of the epididymis were found.
In summary, the ARDmon mouse model shows an intermediate AIS phenotype. Via orchidectomy in combination with androgen replacement therapy, we will now investigate remaining androgen responses in the ARDmon/Y mice and elucidate response elements on which the AR can act in a D-box independent way. Additionally, we generated the first rodent PAIS model with a genetic cause of hypospadias.

Disrupting DNA-binding Domain Dimerization In The Androgen Receptor In Mice Results In a Partial Androgen Insensitivity Model

Introduction: The pituitary gland controls several mechanisms as metabolism, growth, and reproduction, in response to hypothalamic stimuli. The adequate temporal/ spatial expression of transcription factors is mandatory for a normal pituitary development. PROP1 transcription factor is widely known as a key pituitary regulator. Pathogenic variants in the PROP1 gene are the main familial causes of hypopituitarism accounting for about 54% of the cases. Aim: To characterize the expression of genes involved in pituitary cells determination and specification during pituitary high demand period such as sexual maturation compared to adulthood in the Ames mice. Methods: Weight and naso-anal length were measured. Pituitary glands were collected from 5 wild type (WT) and 5 prop1 mutant (Mut) animals with 30 (P30), 40, (P40), and 60 (P60) days after birth. RT-qPCR was performed to analyze the pituitary transcription factors Sox2, Sox3, Hesx1, Otx2, and genes codifying the hormones GH, TSH, LH/ FSH, CGA, and PRL by SYBR® Green (QIAGEN, Valencia, CA). They were normalized by endogenous genes and performed in triplicate. The target genes relative quantification was performed using the mutant related to its age paired wild type as a calibrator and the results were expressed as fold change. Immunofluorescence of SOX2 (ab97959, Abcam, UK) (1: 1600) and pituitary hormones GH, LH, and FSH (1: 250) were done in pituitaries at P60 and its aged paired control, stained with propidium iodide (1: 1000) for nucleus and the secondary antibody Alexa FluorÒ488 (ab150077, Abcam, UK) (1: 1000). Results: All mutant mice presented decreased weight and naso-anal length at the three analyzed periods. The expression of the pituitary stem/ progenitor cell marker Sox2 was increased at P30 and P60 and decreased at P40. Sox3 was increased at P30 and decreased at P40 and P60. Hesx1 was increased at P30 and P60 and decreased at P40. Otx2 was increased at all periods. At P30 the genes codifying pituitary hormones was decreased, except for Tsh and Fsh. At P40 it was observed increased expression of the genes codifying Gh, Lh, Fsh and Prl, while Tsh and Cga expression was reduced. At P60 all pituitary hormones codifying genes presented decreased expression. The immunofluorescence at P60 showed increased expression of SOX2, similar expression of FSH, and decreased expression of GH and LH related to their age paired WT. Conclusion: Despite the absence of PROP1, Hesx1 is downregulated at P40. Pituitary cell subtype differentiation and hormonal production is observed at transcriptional and protein levels, showing that pituitary cells can differentiate and produce hormones in the absence of Prop1. The recovery of pituitary capacity to differentiate in mutant suggests the involvement of another factor in the pituitary cell differentiation pathway that could compensate the lack of Prop1.

PROP1 is not essential for pituitary cells terminal differentiation

The Musashi RNA-binding protein functions as a gatekeeper of cell maturation and maintains stem cell plasticity by regulating the translation of target mRNAs. The adult anterior pituitary tissue expresses a high level of Musashi and also demonstatres a high level of cell plasticity, indicating a Musashi-dependent function in maintaining endocrine homeostasis in the anterior pituitary. Towards understanding the mechanism(s) by which Musashi functions to control cell plasticity, we have identified co-associated proteins necessary for Musashi function. The two Musashi isoforms (Musashi1 and Musashi2) are conserved across species and are structurally characterized by two N-terminal RNA-recognition motifs (RRMs, that associate with specific sequences in the 3' untranslated region of mRNAs targets) and a disordered C-terminus. Musashi uses these domains to selectively repress, or to activate, mRNA translation in a manner specific to target mRNA and cell context. Both Musashi isoforms contain two C-terminal regulatory serine residues that require phosphorylation to enable mRNA translational activation. However, the Musashi proteins do not have inherent mRNA translation function and utilize co-associated protein complexes to mediate mRNA translation. We have previously identified Musashi1 binding protein partners through mass spectrometry. Here, we have identified the protein interactions that are required for Musashi1-dependent translational activation. Knockdown of target protein levels in Xenopus laevis oocytes indicated that the PABP4, CELF2, LSM14A/B, ELAVL1, ELAVL2, ELAVL4, and PUM1 proteins are required for Musashi target mRNA translational activation. We further determined that the effect of LSM14A/B dual knockdown was in fact specific to LSM14B alone. LSM14B is an RNA-binding protein that is required for RNA granule formation, RNA transcript metabolism, and mRNA translation. Studies in mouse oocytes have shown that LSM14B is required to maintain transcript levels of the Musashi1 target Cyclin B1 (Ccnb1) mRNA prior to mRNA translation. In the adult murine pituitary, LSM14B is ubiquitously expressed in both hormone-producing cells and in stem cells as indicated through single cell RNA sequencing. These findings indicate that LSM14B and Musashi1 may function cooperatively to regulate pituitary hormone production and cell differentiation in response to changes in physiological demand.

ESR1 Pathogenic Variant With Incomplete Estrogen Insensitivity

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