ORIGINAL RESEARCH Progesterone Receptor Transcriptome and Cistrome in Decidualized Human Endometrial Stromal Cells Erik C. Mazur,* Yasmin M. Vasquez,* Xilong Li, Ramakrishna Kommagani, Lichun Jiang, Rui Chen, Rainer B. Lanz, Ertug Kovanci, William E. Gibbons, and Francesco J. DeMayo Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 Division of Reproductive Endocrinology and Infertility (E.C.M., E.K., W.E.G.), Department of Obstetrics and Gynecology, Texas Children’s Hospital Pavilion for Women, Department of Molecular and Cellular Biology (Y.M.V., X.L., R.K., R.B.L., F.J.D.), and Department of Molecular and Human Genetics (L.J., R.C.), Baylor College of Medicine, Houston, Texas 77030 Decidualization is a complex process involving cellular proliferation and differentiation of the endometrial stroma that is required to establish and support pregnancy. Progesterone acting via its nuclear receptor, the progesterone receptor (PGR), is a critical regulator of decidualization and is known to interact with certain members of the activator protein-1 (AP-1) family in the regulation of transcription. In this study, we identified the cistrome and transcriptome of PGR and identified the AP-1 factors FOSL2 and JUN to be regulated by PGR and important in the decidualization process. Direct targets of PGR were identified by integrating gene expression data from RNA sequencing with the whole-genome binding profile of PGR determined by chromatin immuno- precipitation followed by deep sequencing (ChIP-seq) in primary human endometrial stromal cells exposed to 17␤-estradiol, medroxyprogesterone acetate, and cAMP to promote in vitro decidu- alization. Ablation of FOSL2 and JUN attenuates the induction of 2 decidual marker genes, IGFBP1 and PRL. ChIP-seq analysis of genomic binding revealed that FOSL2 is bound in proximity to 8586 distinct genes, including nearly 80% of genes bound by PGR. A comprehensive assessment of the PGR-dependent decidual transcriptome integrated with the genomic binding of PGR identified FOSL2 as a potentially important transcriptional coregulator of PGR via direct interaction with regulatory regions of genes actively regulated during decidualization. (Endocrinology 156: 2239–2253, 2015) he endometrium is a dynamic tissue regulated by the the embryo (1). The hallmark of this differentiation or Tovarian steroid hormones, estradiol and progesterone. decidualization, occurs in the stromal compartment of the The endometrium proliferates under the influence of es- endometrium where spindle-shaped fibroblasts transform tradiol produced by growing follicles during the first 2 to plump secretory cells and provide a histiotrophic envi- weeks of the human menstrual cycle. After ovulation, pro- ronment that nourishes the developing embryo while at gesterone from the newly formed corpus luteum drives a the same time limiting the invasiveness of the trophoblast, process of differentiation during which the endometrium a supportive task required for placentation. The decidua becomes competent to receive and support the growth of also creates an interphase where the maternal immunity is ISSN Print 0013-7227 ISSN Online 1945-7170 * E.C.M. and Y.M.V. contributed equally to the study. Printed in U.S.A. Abbreviations: AP-1, activator protein-1; ChIP-seq, chromatin immunoprecipitation fol- Copyright © 2015 by the Endocrine Society lowed by deep sequencing; Co-IP, coimmunoprecipitation; DAVID, Database for Anno- Received July 7, 2014. Accepted March 11, 2015. tation, Visualization, and Integrated Discovery; EPC, estradiol, medroxyprogesterone ac- First Published Online March 17, 2015 etate, and cAMP; FDR, false discovery rate; FOXO1, forkhead box protein O1; GO, Gene Ontology; HESC, human endometrial stromal cell; IGFBP1, IGF binding protein 1; Jak, Janus kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; PGR, progesterone receptor; PRE, progesterone response element; PRL, prolactin; qPCR, quantitative PCR; RNA-seq, RNA se- quencing; siFϩJ, FOSL2 and JUN–targeting small interfering RNA; siFOSL2, FOSL2-targeting small interfering RNA; siJUN, JUN-targeting siRNA; siNT, nontargeting (scrambled) small inter- fering RNA; siPGR, PGR-targeting small interfering RNA; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription; Wnt, wingless-related integration site. doi: 10.1210/en.2014-1566 Endocrinology, June 2015, 156(6):2239–2253 endo.endojournals.org 2239 2240 Mazur et al Decidual PGR Transcriptome and Cistrome Endocrinology, June 2015, 156(6):2239–2253 modulated to tolerate the fetal allograft (2). Because this ponent of the extracellular matrix involved in cell adhe- process is critical to establishing and maintaining preg- sion and migration, mainly through the CRE/AP-1 site nancy, it is likely that defective decidualization underlies located in the proximal region of the promoter in human a certain proportion of infertility in women (3, 4). decidual fibroblasts (15). Last, progesterone has been The fundamental regulator of endometrial stromal cell shown to regulate AP-1 activity in human endometrial decidualization is progesterone, which acts through its nu- adenocarcinoma cells and has been explained as a mech- clear steroid hormone receptor, the progesterone receptor anism by which progesterone inhibits endometrial cancer (PGR) (5). Classically, nuclear steroid hormone receptors cell growth (16, 17). bind DNA directly at specific hormone response elements The aim of this study was to increase our understanding in promoter regions and drive the transcription or repres- of the transcriptional changes that occur during the in sion of particular genes (6). These receptors can also in- vitro decidualization of primary human endometrial stro- Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 teract with DNA indirectly through protein-protein inter- mal cells (HESCs) by taking advantage of recent advances actions with other factors that, in turn, bind DNA. in next-generation sequencing technologies. RNA se- Regardless of direct or indirect DNA binding, nuclear ste- quencing (RNA-seq) offers an important improvement roid hormone receptors associate with complexes of co- over microarrays because of the capture of a higher dy- regulators that are responsible for the events required to namic range of expression levels that more faithfully de- drive or repress transcription (7). Steroid hormones are scribe the robust changes occurring during the secretory known to regulate a variety of seemingly opposing pro- transformation of stromal cells. Second, we aimed to de- cesses, including proliferation and differentiation and, termine the role of PGRs in mediating the expression consequently have different effects in different tissues. changes during decidualization by describing the tran- These context-specific actions are defined by the intracel- scriptome in PGR-silenced cells exposed to a decidual lular milieu and the intrinsic expression of different com- stimulus. Third, we aimed to explore the direct role of ponents of these receptor-coregulator complexes (8). PGRs in this process by describing the whole-genome Identification of relevant PGR coregulators and down- binding profile of PGR during decidualization using chro- stream signaling effectors is critical to the understanding matin immunoprecipitation followed by deep sequencing of the endometrial stromal cell–specific transcriptional (ChIP-seq). With the integration of these robust data sets, changes that occur in the decidualization process. we identified key factors whose expression changes during It has been shown that the activator protein-1 (AP-1) decidualization in a PGR-dependent manner, among them family of transcription factors are involved in the regula- the AP-1 family member FOSL2. Finally, we determined tion of gene expression during cell differentiation and that that a vast majority of PGR-bound genes are also bound there is extensive cross talk between AP-1 and nuclear by FOSL2 and propose that this overlap may underlie a receptors (9–11). AP-1 is composed of homodimers or mechanism of transcriptional cooperation via direct in- heterodimers of members of the Fos and Jun families (FOS, teraction on chromatin during decidualization. The data FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND), and the generated by this study are important to better understand different combinations of these members have been de- the direct and indirect mechanisms underlying PGR action scribed in various contexts in which AP-1 is thought to act. and the involvement of the AP-1 family of transcription The AP-1 member Jun dimerization protein 2 (JDP-2) has factors in the endometrium. been shown to interact directly with the transcription ac- tivation function (AF) domain of PGR and increase hor- mone-dependent PGR-mediated transactivation primar- Materials and Methods ily by stimulating AF-1 activity (12). FOSL1 has been described as a downstream effector of the phosphatidyl- Endometrial stromal cells inositol 3-kinase/AKT signaling pathway responsible for Human endometrial samples were obtained from 6 healthy, reproductive-aged volunteers with regular menstrual cycles and development of trophoblast lineages integral to establish- no history of gynecological malignancies under a human subject ing the maternal-fetal interface, highlighting the impor- protocol approved by the institutional review board of Baylor tance of AP-1 factors in establishment of pregnancy (13). College