Loss of HDAC3 results in nonreceptive endometrium and female infertility

Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus and causes chronic pelvic pain and infertility. However, the exact mechanisms of the pathogenesis of endometriosis-associated infertility are unknown. Epigenetic dysregulation has recently been implicated in infertility. Here, we report a reduction of histone deacetylase 3 (HDAC3) protein amounts in eutopic endometrium of infertile women with endometriosis compared to a control group. To investigate the effect of HDAC3 loss in the uterus, we generated mice with conditional ablation of Hdac3 in progesterone receptor (PGR)–positive cells (Pgrcre/+Hdac3f/f; Hdac3d/d). Loss of Hdac3 in the uterus of mice results in infertility due to implantation failure and decidualization defect. Expression microarray and ChIP-seq analyses identified COL1A1 and COL1A2 as direct targets of HDAC3 in both mice and humans. Reduction of HDAC3 abrogated decidualization in a primary culture of human endometrial stromal cells (hESCs) similar to that observed in infertile patients with endometriosis. Whereas attenuation of HDAC3 resulted in p300 recruitment to Col1a1 and Col1a2 genes in the uterus of mice as well as hESCs, inhibition of p300 permitted hESCs to undergo decidualization. Collectively, we found attenuation of HDAC3 and overexpression of collagen type I in the eutopic endometrium of infertile patients with endometriosis. HDAC3 loss caused a defect of decidualization through the aberrant transcriptional activation of Col1a1 and Col1a2 genes in mice and COL1A1 and COL1A2 genes in humans. Our results suggest that HDAC3 is critical for endometrial receptivity and decidualization.

GREB1 amplifies androgen receptor output in prostate cancer and contributes to antiandrogen resistance

Genomic amplification of the androgen receptor (AR) is an established mechanism of antiandrogen resistance in prostate cancer. Here we show that the magnitude of AR signaling output, independent of AR genomic alteration or expression level, also contributes to antiandrogen resistance, through upregulation of the coactivator GREB1. We demonstrate 100-fold heterogeneity in AR output within cell lines and show that cells with high AR output have reduced sensitivity to enzalutamide. Through transcriptomic and shRNA knockdown studies, together with analysis of clinical datasets, we identify GREB1 as a gene responsible for high AR output. We show that GREB1 is an AR target gene that amplifies AR output by enhancing AR DNA binding and promoting p300 recruitment. GREB1 knockdown in high AR output cells restores enzalutamide sensitivity in vivo. Thus, GREB1 is a candidate driver of enzalutamide resistance through a novel feed forward mechanism.

Embryonic transcription is controlled by maternally defined chromatin state

Histone-modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3- and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300 recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences.

Identification and Characterization By ChIP-Seq Of Genomic Sites Bound By E2A-PBX1 In Acute Lymphoblastic Leukemia Demonstrates Associations With p300 Recruitment, Transcriptionally Active Chromatin and Abundant Transcription

E2A-PBX1 (EP1) is a chimeric oncogenic transcription factor expressed consequent to the 1;19 chromosomal translocation in cases of acute lymphoblastic leukemia (ALL). EP1 can induce transcription of reporter genes and EP1-driven oncogenesis requires direct binding of EP1 with the transcriptional co-activator and histone acetyltransferase p300. Therefore, we hypothesized that EP1 recruits p300 and other co-activators to cis-acting regulatory elements throughout the genome thereby inducing or maintaining transcription of target genes some of which contribute to the neoplastic phenotype. Here we have used chromatin immunoprecipitation followed by next generation DNA sequencing (ChIP-seq) to identify and characterize EP1-bound sites across the genome of the t(1;19)-associated, ALL-derived cell line RCH-ACV. ChIP was performed with an anti-FLAG antibody using sheared chromatin prepared from RCH-ACV cells that stably expressed FLAG-tagged EP1; ChIP from parent RCH-ACV cells not expressing FLAG-EP1 served as a negative control for peak calling. Parallel immunoprecipitations were performed with antibodies for p300 and the chromatin marks H3K4me3, H3K4me1 and H3K27me3. Sequencing of DNA purified from the immunoprecipitated material and of total RNA (RNA-seq) was carried out commercially by BGI whereas bioinformatic analyses were performed in-house. Bioinformatic analysis of data from replicate samples identified 3166 EP1 binding peaks across the RCH-ACV genome (irreproducible discovery rate threshold <0.01). Most EP1 binding sites were located in intronic (1408 sites) or intergenic (1346 sites) regions. Binding site consensus analysis showed overrepresentation of binding motifs for REST, CTCF, MYC, PAX5 and other transcription factors suggesting indirect recruitment of EP1 to DNA mediated by protein-protein interactions. EP1-bound regions were enriched for p300 binding (Figure 1), consistent with the documented importance of p300 recruitment in EP1 oncogenesis. A particular association with H3K4me3 relative to H3K4me1 or H3K27me3 (Figure 2) suggested association with active promoters. Three hundred and forty-two genes had EP1 binding sites within 1000 bp of their transcriptional start sites and these genes were associated with differentially abundant transcription (Figure 3, P<0.001). Querying the online Mammalian Phenotype Ontology tool with genes associated with EP1 binding generated terms that were obviously rich in phenotypes pertaining to B-lymphopoiesis. In summary, our results suggest that EP1 recruits p300 and other co-activators to transcriptionally active chromatin in ALL cells. Results from studies currently underway to confirm the dependency of target gene expression and p300 recruitment upon binding of EP1 at specific binding sites will be presented. Disclosures: No relevant conflicts of interest to declare.