Progesterone Receptor Transcriptome and Cistrome in Decidualized Human Endometrial Stromal Cells

Home , AP3B2, ARPC1B, CCBE1, CCNG2, CRISPLD2, Forkhead box protein O1

ORIGINAL RESEARCH

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 immunoprecipitation followed by deep sequencing (ChIP-seq) in primary human endometrial stromal cells exposed to 17␤-estradiol, medroxyprogesterone acetate, and cAMP to promote in vitro decidualization. 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 sequencing; siFϩJ, FOSL2 and JUN–targeting small interfering RNA; siFOSL2, FOSL2-targeting small interfering RNA; siJUN, JUN-targeting siRNA; siNT, nontargeting (scrambled) small interfering 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 activation function (AF) domain of PGR and increase hormone-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 of Medicine. After receipt of informed consent from The expression of prolactin (PRL), a major secretory prod- participants, an endometrial biopsy was performed during the uct of decidualized endometrium, has been shown to be follicular phase of the menstrual cycle, and endometrial stromal regulated via 2 AP-1 sites in the gene promoter sites, which cells were isolated by enzymatic digestion and filtration, as described previously (4, 18). Stromal cells were cultured in DMEM/ bind specific members of the AP-1 family and not others F12 media with 10% fetal bovine serum, and all experiments (14). Furthermore, it was shown that PGR regulates the were carried out within 4 cell passages. When cells reached ap- promoter activity of fibronectin 1 (FN1), a critical com- proximately 60% confluence, they were transfected with 60 nM doi: 10.1210/en.2014-1566 endo.endojournals.org 2241 scrambled, nontargeting (siNT) or PGR-targeting (siPGR) small rine leukemia virus (Life Technologies) per the manufacturer’s pro- interfering RNA (siRNA) (ON-TARGETplus; GE Dharmacon, tocol. Expression levels of mRNA were determined by qPCR on Lafayette, CO) using Lipofectamine RNAiMAX lipid (Life a QuantStudio 12K Flex Real-Time qPCR system (Life Tech- Technologies) per the manufacturer’s instructions. After 48 nologies) using iTaq Universal SYBR Green Supermix (Bio-Rad hours of transfection exposure, cells were either collected or ex- Laboratories) and primers (Sigma-Aldrich) and normalized to posed to 10 nM 17␤-estradiol, 100 nM medroxyprogesterone 18s RNA (Supplemental Table 1). One-way ANOVA followed acetate, and 1 mM 8-bromo-cAMP (EPC) in Opti-MEM I (Life by a Tukey-Kramer multiple comparisons test was used to com- Technologies) reduced serum media supplemented with 2% pare gene expression levels. stripped fetal bovine serum, antibiotics, and antimycotics for 72 hours. For quantitative PCR experiments, mRNA was isolated ChIP-seq by TRIzol (Life Technologies) extraction per the manufacturer’s PGR, FOSL2, and input ChIP were performed by Active Mo- protocol. For cell viability assays, 103 HESCs were plated per

tif, Inc on HESC cultures established from the 6 endometrial Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 well (96-well plate) 48 hours after transfection with siRNA. Af- biopsy specimens as described previously (18, 22). The model- ter adherence to the well floor, cells were grown in DMEM/F12 based analysis of ChIP-seq (23) algorithm was used to find peaks media with 10% fetal bovine serum (nondecidual) or EPC media by normalizing PGR and FOSL2 ChIP against the input control (decidual). Cell viability was determined using the CellTiter 96 Ϫ with a cutoff of P Ͻ 10 10. Associated genes were called if PGR Non-Radioactive Cell Proliferation Assay (Promega) according or FOSL2 intervals were located within Ϯ10 kb of the gene to the manufacturer’s instructions. boundaries. Analyses of gene distribution and enriched motifs were performed using Cistrome (http://cistrome.org/ap/) (24). RNA-seq Gene functional analysis was performed using the Database for RNA was purified from HESC cultures established from 3 Annotation, Visualization, and Integrated Discovery (DAVID) independent biopsy samples and treated as described above for (http://david.abcc.ncifcrf.gov/) (25, 26). The PGR ChIP-seq data RNA-seq analysis using the Ambion RiboPure Kit (Life Tech- set was also used in 2 additional publications, one in which a nologies). RNA-seq was performed for each individual patient comparative analysis of ancient mammalian transposable el- sample. Raw reads were mapped to human genome hg19 and ements involved in decidualization was performed and the splice junction sites using Bowtie (v0.12.7) (19) and TopHat second one in which the requirement of forkhead box protein (v2.0.0) (20) with the strand-specific model that matches our O1 (FOXO1) for PGR binding in decidualization was evalu- dUTP library construction protocol. The human annotation ated (27, 28). file was downloaded from UCSC Genome Browser (http:// genome.ucsc.edu/). Read counts to each gene were calculated by HTSeq (http://www.huber.embl.de/users/anders/HTSeq/doc/ ChIP-seq validation overview.html) using the default model. Differential gene expres- ChIP-seq PGR intervals on FOSL2 and JUN were confirmed sion was analyzed with R (v2.14.0; http://www.R-project.org) and by ChIP-qPCR. HESCs were grown to 80% confluence before the Bioconductor edgeR package (edgeR_2.4.6) (21). With exposure to a decidual stimulus (EPC). After 72 hours of expo- edgeR, we fit a negative binomial generalized log-linear model sure, ChIP was performed using the SimpleChIP Enzymatic to the read counts for each gene by accounting for both patient Chromatin IP Kit (Cell Signaling Technology) following the and treatment in the design. Gene-wise statistical tests were manufacturer’s instructions. In brief, proteins were cross-linked conducted to identify genes that have consistent changes in to DNA with formaldehyde, and the cells were treated with mi- response to treatment across 3 individuals. A false discovery crococcal nuclease and mechanical shearing to digest DNA and rate (FDR) of 0.05 was used as the cutoff for significant dif- break nuclear membranes. This sheared chromatin was immu- ferential expression. noprecipitated with antibodies to PGR and IgG (H-190/sc-7208 Gene expression changes observed by RNA-seq were vali- and sc-2027, Santa Cruz Biotechnology, Inc) (Table 1). Primers dated in replicate experiments by real-time RT-quantitative for SYBR Green RT-qPCR were designed to query the PGR in- PCR(qPCR). mRNA was isolated by TRIzol (Life Technologies) tervals on FOSL2 and JUN as determined by the ChIP-seq (Sup- extraction and reverse transcribed into cDNA with Moloney mu- plemental Table 1). Two untranslated regions were queried as a

Table 1. Antibody Table

Manufacturer, Catalog No, and/or Name Peptide/ of Individual Species Raised Protein Antigen Sequence Name of Providing the in (Monoclonal Target (If Known) Antibody Antibody or Polyclonal) Dilution Used PGR Amino acids 375–564 PR (H-190) Santa Cruz, sc-7208 Rabbit polyclonal 4 ␮g/immunoprecipitation of PGR of human reaction origin Fra-2 (FOSL2) Epitope mapping at the Fra2 (Q-20) Santa Cruz, sc-604 Rabbit polyclonal 4 ␮g/immunoprecipitation N terminus of Fra-2 reaction of human origin Normal rabbit Normal rabbit Santa Cruz, sc-2027 Rabbit 4 ␮g/immunoprecipitation IgG IgG reaction 2242 Mazur et al Decidual PGR Transcriptome and Cistrome Endocrinology, June 2015, 156(6):2239–2253

negative control (Human ChIP Control qPCR Primer Set, Active mM EGTA [pH 7.0], and 2 mM MgCl2) for 30 minutes. Fix- Motif). qPCR was used to compare PGR-immunoprecipitated to ative was removed, and cells were washed 3 times with PEM IgG-precipitated chromatin. The presence of putative hormone alone. Cell delineation was determined by staining with HCS response elements for PGR within the FOSL2 and JUN intervals CellMask Blue (catalog no. H32720; Life Technologies) di- was determined using the transcription factor binding profile luted 1:100 000 in 0.1% Triton X-100 in PEM for 15 minutes database, JASPAR (http://jaspar.genereg.net/). and subsequently washed 3 times with PEM. Coverslips were mounted on slides with SlowFade Gold (catalog no. S-2828; Analysis of AP-1 factors Molecular Probes). Images were acquired using a GE Health- qPCR was used to investigate the effect of silencing FOSL2 care DeltaVision Image Restoration microscope with a 40x/ and JUN in HESCs exposed to a decidual stimulus. As described 0.95na objective. Optical sections (0.35-mm; z-stacks) were above, HESCs were transfected with siRNA targeting FOSL2, taken and deconvolved using softWoRx software, and maximum intensity was projected.

JUN, or a dual knockdown of FOSL2 and JUN. Transfected cells Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 were exposed to the same decidual stimulus, and mRNA was isolated by TRIzol extraction. cDNA construction was performed, and mRNA levels were determined by qPCR using an Results SYBR Green protocol and primers designed using the Primer- BLAST suite at the National Center for Biotechnology Infor- Transcriptome of decidualizing HESCs mation (NCBI) and normalized to 18s RNA (Supplemental Table 1). To identify the mRNA profile change during HESC decidualization, RNA-seq was performed in HESCs trans- Coimmunoprecipitation (Co-IP) of FOSL2 and JUN fected with siNT with and without a hormone stimulus. A Co-IP of FOSL2 and JUN was performed using the Universal total of 4061 genes were differentially regulated between Magnetic Co-IP kit (catalog no. 54002; Active Motif) as per the nondecidual and decidual HESCs (Supplemental Table 2). manufacturer’s instructions. In brief, HESCs were grown to 70% Table 2 summarizes the top pathways enriched in the func- confluence and treated with EPC for 72 hours to stimulate de- tional pathway analysis performed with DAVID bioinfor- cidualization as described above. One hour before harvest, cells were treated with fresh media and hormones. Cell collection, matics resources (25, 26). Enrichment was seen in path- whole-cell extraction, and quantification were performed as per ways for cell communication, cell cycle, and metabolism, the manufacturer’s instructions. Co-IP was performed with 400 consistent with the expected processes in cells undergoing ␮ ␮ g of protein and 1 g of IgG, FOSL2, and JUN antibodies decidualization. Decidualizing HESCs are characterized (catalog nos. sc-2027, sc-604, and sc-1694; Santa Cruz Biotech- by cell enlargement, nuclear rounding, expansion of the nology) for 3 hours. Magnetic beads were incubated with the antibody/extract mixtures (1 hour, 4°C) and subsequently Golgi/endoplasmic reticulum, and accumulation of glyco- washed 4 times with complete/Co-IP wash buffer. Bead pellets gen and lipid in the cytoplasm. These cells are active se- were resuspended in 2ϫ reducing loading buffer (catalog no. cretory cells with secreted products commonly used as ␤ 161–0737; Bio-Rad Laboratories) containing 10% -mercap- markers of decidualization. Most commonly, IGF binding toethanol and boiled for 5 minutes at 90°C before electropho- protein-1 (IGFBP-1) and PRL have been monitored as mo- resis separation in Bis-Tris NuPAGE 4%–12% gels (1.0 mm, 10-well; catalog no. NP0321BOX, Novex; Life Technologies). lecular markers for the decidualization process, and in our Proteins were transferred to polyvinylidene difluoride mem- data, IGFBP1 and PRL are 2 of the most highly induced branes (EMD Millipore) in transfer buffer (25 mM Tris, 192 mM genes with exposure to EPC. These data confirmed the glycine, and 20% methanol) (Life Technologies). polyvinylidene induction of cytokines (IL11), growth factors (heparin- difluoride membranes were subsequently blocked with 5% blot- binding epidermal growth factor [HBEGF]), neuropep- ting grade nonfat milk (catalog no. 170–6404; Bio-Rad) in PBS containing 0.1% Tween 20 (PBST) for 1 hour at room temper- tides (somatostatin), and critical transcription factors ature. Membranes were probed with antibodies for FOSL2 and (FOXO1), which are expressed in decidualizing stro- JUN overnight at 4°C in 5% blotting grade nonfat milk in PBST. mal cells at high levels and thought to amplify and prop- Blotted membranes were subsequently washed 3 times with agate the decidual process (1). Furthermore, decidual- PBST and incubated for 1 hour (room temperature) with anti- izing HESCs contain surface projections that extend rabbit peroxidase secondary antibody. Blots were washed 3 times with PBST and subsequently 3 times with PBS. Luminol- both into the extracellular matrix and into adjacent based detection of bands on film was performed using the Am- cells, and adherens junctions are found between adja- ersham ECL Western Blotting System (GE Healthcare) as per the cent cells. Data confirmed expression changes in manufacturer’s instructions. decorin, laminins, type IV collagen, and fibronectin, which are known to be involved in the remodeling of Imaging of HESC morphology decidual cells. Among the signaling pathways enriched HESCs were grown on coverslips and treated with EPC for ␤ 3 days to stimulate a decidual response as described above. were TGF- , Janus kinase (Jak)-signal transducer and Cells were washed with cold PBS once and fixed with 4% activator of transcription (STAT), and wingless-related formaldehyde in PEM buffer (potassium PIPES [pH 6.8], 5 integration site (Wnt) pathways. doi: 10.1210/en.2014-1566 endo.endojournals.org 2243

Table 2. DAVID Functional Analysis Using KEGG Pathways for Genes With Differential Expression (FDR Ͻ 0.05) by RNA-Seq in HESCs Before and After Treatment With a Decidual Stimulus (EPC)

Description, (KEGG hsa Identification No.) Genes Cell communication Focal adhesion (4510) PDGFB, TLN2, BCAR1, VTN, VCL, ACTG1, PDGFC, PDGFD, PAK1, COL11A1, RAPGEF1, SHC2, AKT3, ACTN4, MYLK3, PIK3CD, FLNC, PPP1CB, FLNB, VEGFC, CCND1, CCND2, RASGRF1, JUN, VEGFA, PDGFRA, MAPK8, CAV2, CAV1, DIAPH1, TNC, COL3A1, ITGA11, ITGB3, PXN, LAMB3, LAMB2, DOCK1, RAC2, ITGB8, ITGAV, THBS1, THBS2, PIK3R1, FN1, COL4A4, ITGA1, IGF1, ITGA2,

MYL12B, ITGA3, MAPK10, HGF, MYL12A, ITGA4, CAPN2, BIRC3, COL5A2, Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 COL5A1, COL4A6, LAMA2, LAMA1, ITGA9, ITGA6, FYN, ITGA8, ITGA7, MYLK Regulation of actin FGF5, ENAH, PDGFB, FGF9, BCAR1, IQGAP3, IQGAP2, INSRR, VCL, ACTG1, GSN, cytoskeleton (4810) PDGFC, PDGFD, PAK1, FGF1, ACTN4, LIMK1, MYLK3, PIK3CD, MYH9, ARHGEF12, PPP1CB, ARPC1B, CHRM2, RRAS2, PDGFRA, TMSB4X, FGFR1, SSH1, DIAPH1, DIAPH2, DIAPH3, ITGA11, ITGB3, GNG12, PXN, DOCK1, EZR, RAC2, ITGB8, ITGAV, PIK3R1, FN1, ITGA1, ITGA2, IGF2, MYL12B, ITGA3, MYL12A, ITGA4, ITGA9, ITGA6, ITGA8, ITGA7, PIP4K2A, MYLK, F2R, MYH10 Pathways in cancer (5210) E2F1, FGF5, E2F3, PDGFB, FGF9, STAT5A, PPARG, STAT5B, ARNT2, TGFB3, FOXO1, MMP1, GLI1, TGFB2, WNT2, CCNE2, WNT4, CDKN2B, SLC2A1, RALA, CSF3R, RARB, FGF1, MYC, AKT3, PIK3CD, TP53, LEF1, CDK6, CTNNA1, DAPK3, CDK2, VEGFC, CCND1, HIF1A, JUN, VEGFA, PDGFRA, MDM2, MAPK8, WNT11, WNT5A, BID, FGFR1, WNT5B, NFKBIA, BCL2L1, ZBTB16, KIT, TCF7L1, SUFU, LAMB3, LAMB2, RAC2, ITGAV, RUNX1, TRAF4, PIK3R1, FN1, COL4A4, BMP4, CEBPA, FZD8, TCF7, IL6, MSH2, SMAD3, IGF1, BRCA2, ITGA2, ITGA3, BIRC5, FZD2, HGF, MAPK10, STAT1, BIRC3, COL4A6, STAT3, FZD7, RALGDS, WNT2B, LAMA2, LAMA1, RASSF5, CDKN1A, HSP90B1, HDAC2, ITGA6, PLCG1, ETS1, NTRK1, BAX, PTCH1, ABL1 Cell cycle (110) E2F1, E2F3, TGFB3, TTK, PKMYT1, PTTG1, TGFB2, CCNE2, CDC45, MCM7, CDKN2B, BUB1, MYC, CCNA2, CDK1, CDC6, RBL1, TP53, SMAD3, CDK6, CDC20, ESPL1, MCM2, CDC25C, MCM3, MCM4, MCM5, WEE1, CDK2, CDC25B, CCNB1, CDKN1C, CDKN1A, CCND1, MAD2L1, YWHAH, CCNB2, HDAC2, CCND2, PLK1, PCNA, BUB1B, MDM2, ABL1 p53 signaling (4115) BID, ZMAT3, RRM2B, PMAIP1, CCNG2, GTSE1, SESN3, CCNE2, TP53I3, SERPINE1, THBS1, CDK1, TP53, IGF1, CDK6, CDK2, CCNB1, CDKN1A, CCND1, PPM1D, TNFRSF10B, CCNB2, CCND2, BBC3, BAX, RRM2, DDB2, MDM2 Metabolism N-Glycan biosynthesis (510) B4GALT1, ST6GAL1, GANAB, MAN1A2, FUT8, ALG1, ALG2, ALG3, ALG5, MOGS, ALG9, LOC151162, STT3A, RPN1, DPM3, RPN2, MGAT5, DDOST Arginine and proline SAT1, ODC1, ALDH18A1, ASS1, MAOA, MAOB, ALDH3A2, CKB, GLUL, PYCR2, metabolism (330) P4HA2, P4HA1, GLS, P4HA3, ALDH2, OAT, SMS Glycosaminoglycan ARSB, SGSH, HGSNAT, GNS, NAGLU, HYAL3, HPSE, GUSB, GLB1 degradation (531) Lysosome (4142) ARSB, HGSNAT, SGSH, NAGLU, GM2A, ARSG, ATP6AP1, LGMN, ACP5, PPT1, CTSL1, ASAH1, GLB1, SLC11A2, AP1S1, CD68, GNPTAB, AP3B2, ATP6V0D2, LIPA, GUSB, MANBA, GNS, LAMP2, CTSK, NPC1, GLA, SMPD1, ARSA, GAA, CTSB P4-mediated oocyte maturation ADCY3, CDK1, ADCY4, PIK3CD, IGF1, PDE3B, PKMYT1, IGF2, MAPK10, CDC25C, (4914) PPP1CB, CDK2, PRKX, CDC25B, CCNB1, RPS6KA6, CCNB2, MAD2L1, PLK1, BUB1, MAPK8, CCNA2, PIK3R1, AKT3 Signal transduction TGF-␤ signaling (4350) BMP4, PPP2R1B, NOG, LTBP1, SMAD7, RBL1, BMPR2, TGFB3, SMAD3, DCN, SMAD1, TGFB2, INHBA, CDKN2B, ID2, INHBE, ID1, LEFTY2, ID4, SMURF2, ID3, THBS1, THBS2, MYC Jak-STAT signaling (4630) IL6ST, STAT5A, LEPR, IL19, STAT5B, CNTFR, BCL2L1, IL15, IL7R, SPRY4, IL11, LIF, SPRY2, SPRY1, STAT4, IL4R, SPRED2, CSF3R, CSF2RB, SPRED1, PRL, MYC, AKT3, IFNGR1, PIK3R1, GHR, IL6, SOCS1, PIK3CD, LIFR, IL24, STAT1, IL11RA, STAT3, IL20, CCND1, CCND2, JAK2 Wnt signaling (4310) WNT5A, NKD1, WNT5B, MMP7, CXXC4, PRKX, TCF7L1, WNT2, WNT4, RAC2, PLCB1, MYC, CAMK2A, FOSL1, PPP2R1B, FZD8, TBL1XR1, TCF7, VANGL2, TP53, SMAD3, LEF1, MAPK10, FZD2, PORCN, FZD7, WNT2B, CCND1, DKK1, PRICKLE1, CCND2, JUN, PRICKLE2, SFRP4, WNT11, MAPK8, TBL1X 2244 Mazur et al Decidual PGR Transcriptome and Cistrome Endocrinology, June 2015, 156(6):2239–2253

To evaluate the role of PGR in the regulation of decid- (Mothers against decapentaplegic homolog 4 [SMAD4] ual genes, HESCs were transfected with siRNA targeting and the transcription factor specificity protein 1 [SP1]) do PGR before decidual stimulus (siPGR EPC). A total of not exhibit changes in expression with decidualization 4960 genes were differentially regulated with PGR knock- (siNT ϩ EPC) but are down-regulated upon silencing of down compared with those for siNT EPC (Supplemental PGR (siPGR ϩ EPC). Table 2). Of the genes, 52% that were differentially regulated during decidualization were also regulated when Identification of PGR global genomic binding sites siPGR EPC cells were compared with siNT EPC cells. in HESCs using ChIP-seq Pathway analysis by Kyoto Encyclopedia of Genes and To identify direct targets of PGR in HESC decidualiza- Genomes (KEGG) functional groups revealed distinct bi- tion, PGR association with DNA was assessed by ChIP- ological pathways for the 3 resulting gene groups: genes seq. The binding of PGR was defined to 8690 intervals. Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 changing with decidual stimulus (siNT vehicle vs siNT and 5091 of these intervals were located within 10 kb of EPC), genes whose expression changed only with siPGR gene boundaries of 3478 genes (Supplemental Table 3). transfection (siNT EPC vs siPGR EPC), and genes with CEAS enrichment analysis showed no significant enrich- expression changes both during decidualization (siNT ve- ments for specific genomic boundaries relative to those for hicle vs siNT EPC) and with PGR knockdown (siNT EPC the genome reference (Figure 2A) or the promoter region vs siPGR EPC). Distinct biological processes were evident of genes (Figure 2B). Motif analysis of PGR intervals using in genes differentially expressed with decidualization, the SeqPos tool in the Cistrome data analysis platform (24) genes with expression changes with PGR knockdown, and revealed enrichment in binding sequences for the steroid genes both differentially regulated with decidualization nuclear hormone receptors (including PGR), forkhead do- and PGR knockdown. Results from the DAVID analysis main, homeobox, interferon regulatory factor, and leu- suggest that the TGF-␤ pathway and cell cycle regulation cine zipper factors (Figure 2C). The nuclear hormone re- are differentially regulated with decidualization alone, sponse element is the most highly enriched binding motif metabolic pathways are enriched with PGR knockdown in the PGR cistrome, suggesting that PGR is frequently alone, and adhesion, cytoskeleton, Jak-STAT, and Wnt bound to the progesterone response element (PRE) during pathways are both PGR-regulated and enriched with de- HESC decidualization and near regions recognized by fac- cidualization (Figure 1A). In the analysis of the PGR tran- tors such as FOXO1, HOXA11, IRF4, and AP-1 family scriptome, genes fall in 2 distinct categories with very clear members. decidual and nondecidual biology. The EPC-regulated Overall, 695 genes were found to be regulated during and siPGR-regulated genes enrich for Gene Ontology decidualization, to be differentially regulated with PGR (GO) terms including regulation of proliferation, vascular knockdown, and to contain bound PGR during decidual- development, apoptosis, migration, adhesion, and cyto- ization (Figure 3A). DAVID analysis revealed that the di- skeletal organization (Supplemental Table 3). These pro- rect PGR target genes enriched biological themes for cesses are hallmarks of the robust transformation stromal regulation of cell motion, vascular development, and in- fibroblasts undergo during decidualization. In contrast, tracellular cascade (Table 3). Among the known direct the siPGR-regulated genes that are not regulated with EPC PGR target genes involved in decidualization, we observed decidualization enrich for terms related to protein modi- PGR binding and differential regulation in several genes fication, catabolism, and localization (Supplemental such as FK506 binding protein 5 (FKBP5), heart- and neu- Table 4). ral crest derivatives-expressed protein 2 (HAND2), and Representative examples of qPCR validation of gene cysteine-rich angiogenic inducer 61 (CYR61) (29, 30). expression changes detected by RNA-seq are shown in ChIP-seq did not identify binding of PGR to the promoter Figure 1B. Frizzled-10 (FZD10) is induced with exposure of IGFBP1, a finding that was inconsistent with previous to EPC (siNT ϩ EPC) but is not dependent on PGR as reports that demonstrated progesterone-responsive regu- siPGR does not change the gene induction with decidual- lation of promoter activity (31). IGFBP1 expression was ization (siPGR ϩ EPC). Similarly, the gene encoding the significantly attenuated with PGR silencing in decidual- Krüppel-like transcription factor GLIS family zinc finger izing HESCs. Similarly, binding of PGR was not observed 2(GLIS2) is down-regulated with decidualization in a proximal to the PRL gene but instead was located 25 kb PGR-independent fashion. Signal transducer and activa- downstream. Consistent with previous reports, we ob- tor of transcription 3 (STAT3) and Dikkopf-related pro- served differential regulation of FGF9, WNT2, CNR1, tein 1 (DKK1) are PGR dependent. Induction of STAT3 ARNT2, and GATA6 with siPGR (5). However, only and DKK1 with decidualization (siNT ϩ EPC) is blunted ARNT2 was bound by PGR within 10 kb of official gene with knockdown of PGR (siPGR ϩ EPC). Certain genes annotations. Other known progesterone-regulated targets doi: 10.1210/en.2014-1566 endo.endojournals.org 2245 Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021

Figure 1. A, Venn diagram of differentially expressed genes in HESCs by RNA-seq. RNA-Seq: EPC is a comparison of nontargeting siRNA- transfected HESCs treated with vehicle or EPC. RNA-Seq: siPGR is a comparison of cells exposed to nontargeting siRNA with those exposed to siRNA targeting PGR (both after treatment with EPC). DAVID functional analysis of KEGG pathways in genes differentially expressed with EPC, differentially expressed with siPGR transfection, and differentially expressed with both EPC and siPGR transfection is shown. B, Gene expression validation by RT-qPCR and normalization with 18s rRNA. Data are based on 3 independent experiments. Error bars represent SEM. *, P Ͻ .05, **, P Ͻ .01, n.s., not significant. such as the ZEB1, KLF15, HOXA10, and PTGS1 were Role of AP-1 in HESC decidualization also found to have PGR binding sites and undergo differ- The genes differentially regulated with decidualization ential regulation with siPGR (Supplemental Table 2) and PGR silencing while containing bound PGR during (32–36). decidualization included 2 members of the AP-1 family of 2246 Mazur et al Decidual PGR Transcriptome and Cistrome Endocrinology, June 2015, 156(6):2239–2253

with PGR knockdown, and these changes in gene expression were confirmed by qPCR (Figure 3B). The decidual and PGR-dependent expression of other members of the AP-1 family (FOS, FOSB, FOSL1, JUNB, and JUND) was also evaluated (Sup- plemental Figure 1). This analysis revealed that whereas FOSL1 undergoes hormone dependent down-regulation

during decidualization, other mem- Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 bers of the AP-1 family are not differentially regulated. Furthermore, the expression of these members is unaf- fected by PGR knockdown. This evidence collectively highlights the specificity and importance of FOSL2 and JUN in HESC decidualization. ChIP-seq analysis revealed PGR binding in 2 regions of FOSL2, im- mediately upstream and approxi- mately 5 kb downstream of the transcriptional start site. PGR binding was also seen upstream of JUN, and these intervals of PGR binding were confirmed by ChIP-qPCR analysis (Figure 4). All 3 of these intervals of DNA contain multiple putative sequences for PGR binding, as determined by in silico analysis (Supple- mental Table 5). The association of FOSL2 and JUN during decidualization was evaluated by performing Co-IP in decidualized HESCs. Immunoprecipi- tation with either FOSL2 or JUN antibody was able to precipitate both factors (Figure 5A), suggesting that FOSL2 and JUN may interact as a dimer in decidual HESCs. We followed this analysis with an evalua- tion of the role of FOSL2 and JUN in proliferation and decidualization. HESCs were transfected with scram- Figure 2. ChIP-seq analysis of PGR cistrome. A, CEAS enrichment analysis of PGR intervals bled siRNA (siNT) and FOSL2-tar- compared with the expected genomic distribution. B, CEAS enrichment analysis of PGR intervals on promoter regions. C, Chromosome location coordinates of PGR binding intervals were geting siRNA (siFOSL2), JUN-tar- analyzed with the SeqPos tool in Cistrome. The 5 most highly enriched consensus binding motifs geting siRNA (siJUN), and FOSL2 ϩ Ͻ Ϫ30 in PGR intervals by Cistrome analysis, P 10 . UTR, untranslated region. JUN-targeting siRNA (siFϩJ) and cultured in standard growth media transcription factors. Fos-like antigen 2 (FOSL2 or FRA2) and EPC media. At day 0, the cell proliferation and via- and jun proto-oncogene (JUN) were each up-regulated bility assay revealed no differences between the groups for with exposure to a decidual stimulus and down-regulated either treatment. At day 3, we observed an increase in the doi: 10.1210/en.2014-1566 endo.endojournals.org 2247

the UCSC Genome Browser for the TGFBR3 and FKBP5 gene loci.

Discussion

PGR is essential for inducing endometrial stromal cell decidualization, a requirement for the establishment and maintenance of pregnancy (37).

In this study, we examined the PGR Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 cistrome and transcriptome during decidualization to better define the molecular mechanisms of PGR-mediated stromal cell function. RNA- seq was used to obtain global expression profiles of stromal cells with and without exposure to a decidualization stimulus of EPC. We present the transcriptome of EPC-treated HESCs transfected with nontargeting siRNA compared with those of 2 groups: ve- Figure 3. Direct targets of PGR in HESC decidualization. A, Venn diagram comparing the genes with annotation of PGR genomic binding within 10 kb of the genomic boundary and the genes hicle-treated HESCs transfected with regulated in HESCs transfected with siRNA targeting PGR before EPC treatment. B, Gene nontargeting siRNA and EPC-treated expression validation of PGR knockdown and direct gene targets by RT-qPCR and normalization HESCs transfected with PGR-tar- Ͻ with 18s rRNA. Data are based on 3 independent experiments. Error bars represent SEM. *, P geting siRNA. One limitation of this .05. D0, day 0; D3, day 3. strategy is the secondary effects of transfection on gene expression proliferation of cells transfected with siFOSL2 only in the changes that occur during decidual- nondecidual conditions. In addition, we observed a de- ization. However, despite these limitations, the robust crease in proliferation in the siJUN and siFϩJ groups un- power of this RNA-seq strategy was able to provide der decidualizing conditions (Figure 5B). greater sensitivity than standard expression arrays in After 6 days of hormone stimulus, HESCs transfected which a similar transfection and treatment strategy was with siNT displayed a decidual, rounded morphology. used (3). HESCs transfected with siFOSL2, siJUN, and siFϩJ dis- We subsequently compared the genes regulated in de- played highly flattened and elongated fibroblastic cell cidualization (siNT vehicle vs siNT EPC) and the PGR- morphology indicative of an absent decidual transforma- dependent genes (siNT EPC and siPGR EPC) to discern tion (Figure 5C). qPCR analysis confirmed a lack of genes that were regulated during decidualization in a FOSL2 and JUN induction when cells were transfected PGR-dependent manner. In this comparison, we observed with targeting siRNA. In addition, inductions of IGFBP1 that a subset of PGR-dependent genes do not overlap with and PRL were blunted by knockdown of FOSL2 and JUN the decidual gene set (2830 genes). GO analysis revealed (Figure 5D). that the PGR-only transcriptional profile constitutes a When genomic DNA binding of FOSL2 was assessed by nondecidual biology. It has been shown that unliganded ChIP-seq in decidualizing HESCs, 24 298 intervals of PGR is able to regulate expression of genes by binding to FOSL2-bound DNA were found, representing proximity genomic sites with repressive protein complexes to silence (Ϯ10 kb) to 8586 distinct genes. FOSL2 was thus found to gene expression in T47D breast cancer cells (38). Al- bind more than twice as many genes as PGR. Furthermore, though expression and transcriptional activity of PGR ap- nearly 80% (2709 of 3477) of PGR-bound genes were pear to peak in decidual conditions when hormone is pres- likewise bound by FOSL2. In examination of the genes ent, it is possible that in nondecidual conditions PGR is targeted by both PGR and FOSL2, a large degree of in- engaged in transcriptional repression. Ablation of PGR terval overlap was observed. Examples are shown in Fig- would consequently result in changes in gene expression ure 6, in which ChIP-seq binding intervals are displayed on in the basal state of stromal fibroblasts before hormone 2248 Mazur et al Decidual PGR Transcriptome and Cistrome Endocrinology, June 2015, 156(6):2239–2253

Table 3. DAVID Functional Pathway Analysis of the 695 Genes Bound by PGR And Differentially Expressed With Decidualization and siPGR

Pathway Genes P Value/FDR Regulation of cell motion (GO: DLC1, RTN4, IL6ST, STAT5B, ITGB3, TPM1, VCL, TRIB1, NISCH, S1PR1, TEK, 3.62EϪ12/6.47EϪ09 0051270) SCARB1, THBS1, INSR, PIK3R1, COL18A1, IRS2, ACTN4, PDPN, ITGA2, PTPRU, LAMA2, CDH13, LAMA1, LAMA4, ETS1, F3, RRAS2, PDGFRA, TGFBR3, JAK2, HDAC9, F2R Vasculature development (GO: RTN4, ZFAND5, COL3A1, TIPARP, ENPEP, ELK3, CDH2, WT1, SEMA5A, SHB, APOB, 1.28EϪ11/2.29EϪ08 0001944) S1PR1, MYOCD, CTGF, HAND2, CCBE1, SEMA3C, FGF1, THBS1, CYR61, RECK, COL18A1, PTPRJ, PLAT, PDPN, MYO1E, MMP19, COL15A1, ARHGAP24, COL5A1, CDH13, SH2D2A, LAMA4, ID1, JUN, TGFBR3, ENG Intracellular signaling cascade ADCY3, IL6ST, LHCGR, STAT5B, TNFSF15, IQGAP2, TLR4, LPAR1, RGL1, PRKAR2B, 9.26EϪ09/1.65EϪ05 Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021 (GO: 0007242) ARHGAP6, HTR1B, MAP3K4, NOD1, S1PR1, NISCH, CTGF, MDFIC, RASL10B, RAB23, SPRED2, GNG2, DLG5, CALCRL, SHC3, AGAP1, PLCB1, FGF1, INSR, FRS2, DDAH1, DHCR24, MYO6, TNIK, LIMK1, ARHGEF7, SOCS6, ARHGEF12, UBE2C, STK3, FOXN3, MAP4K3, TNS3, MAP4K4, FMN2, CHRM2, RRAS2, RIN2, ROR2, MAPK7, KSR1, LRRK1, CARHSP1, BLM, DUSP10, NFKBIA, MAPKAPK2, TRIB1, SQSTM1, RASGRP1, PKD2, SH2B3, TNFRSF19, RHOBTB1, THBS1, KNDC1, PIK3R1, FGD4, ARHGDIB, RAB8B, TAOK3, ITGA1, DGKH, NDC80, NPR3, DGKI, RGNEF, RACGAP1, HOMER1, STAT3, TRAF3IP2, SH3BP5, RASL11B, CDH13, DUSP1, FYN, TGFBR3, JAK2, GRK5, CIT, F2R Cell adhesion (GO: 0007155) DLC1, CLSTN2, TLN2, CLSTN1, LMO7, EDIL3, VCL, ARHGAP6, S1PR1, WISP1, 4.72EϪ08/8.43EϪ05 CTGF, ROBO2, DLG5, COL11A1, CYR61, PDPN, MGP, PTPRU, PCDH7, ROR2, VCAN, ADAM12, EPDR1, TNC, COL3A1, ITGA11, NEDD9, ITGB3, CDH2, PKD1L1, ITGBL1, SEMA5A, SORBS1, COL7A1, KAL1, TEK, PKD2, SCARB1, THBS1, COL18A1, SVEP1, GMDS, PPFIBP1, ITGA1, COL15A1, ITGA2, NID1, ITGA3, COL5A1, PCDH18, LAMA2, LAMA1, CDH13, LAMA4, COL14A1, ITGA6, PDZD2, ENG, NTM Enzyme-linked receptor protein ZFAND5, LTBP2, IL6ST, COL3A1, TIPARP, STAT5B, GREM2, SLC2A8, EPHB6, 1.98EϪ07/3.53EϪ04 signaling pathway (GO: SORBS1, CTGF, TEK, PDGFC, SHC3, NRG1, FGF1, FRS2, INSR, PIK3R1, PTPRJ, 0007167) PLAT, IRS2, NIN, MYO1E, PTPRU, STAT3, GRB10, ID1, JUN, PDGFRA, ROR1, TGFBR3, SORT1, ROR2, JAK2, ENG Response to organic substance ADCY3, ATP6V0E1, IL6ST, LHCGR, ARNT2, STAT5B, SNCA, TLR4, SLC2A8, 1.52EϪ06/0.002708 (GO: 0010033) PRKAR2B, APOB, HTR1B, GNG2, INSR, CYR61, STS, IRS2, MGP, PTPRU, GRB10, JUN, PDGFRA, SORT1, DERL1, PANX1, ENPP1, COL3A1, NFKBIA, TIMP4, BCL2L1, C1S, GLRX2, TRIB1, IRAK3, SORBS1, HSPA2, PLIN2, PEMT, SCARB1, THBS1, PIK3R1, MAP1B, ITGA2, STAT3, HDAC4, CDH13, DUSP1, ID1, FYN, FABP4, TGFBR3, JAK2, IGFBP2, HDAC9, DNAJB6, F2R Extracellular structure RECK, COL18A1, MYO6, TNC, ADAMTSL4, ELN, COL3A1, MAP1B, NID1, DCN, 5.89EϪ06/0.01051 organization (GO: 0043062) CDH2, CACNB4, COL5A2, COL5A1, COL14A1, CRISPLD2, PDGFRA, COL11A1, ENG, F2R, CYR61 Protein kinase cascade (GO: IL6ST, STAT5B, DUSP10, TNFSF15, NFKBIA, TLR4, MAPKAPK2, LPAR1, TRIB1, 9.09E–06/0.01622 0007243) MAP3K4, MDFIC, SPRED2, PKD2, TNFRSF19, THBS1, FGF1, INSR, FRS2, PIK3R1, FGD4, TNIK, TAOK3, ITGA1, SOCS6, STK3, STAT3, MAP4K3, MAP4K4, FYN, ROR2, TGFBR3, JAK2, MAPK7, F2R Response to wounding (GO: TPST1, MASP1, TNC, STAT5B, COL3A1, AFAP1L2, TLR4, C1R, ITGB3, C1S, ELK3, 1.09EϪ05/0.019512 0009611) LMAN1, TPM1, TNFRSF1B, HMCN1, NOD1, CTGF, SCARB1, THBS1, NRG1, NOX4, PLAT, KLF6, LIPA, TNFSF4, PDPN, MAP1B, ITGA2, COL5A1, STAT3, HDAC4, PLSCR1, PLSCR4, F3, PDGFRA, C1RL, TFPI, NFE2L1, VCAN, JAK2, HDAC9, ENG, F2R Cellular response to hormone ADCY3, IRS2, ENPP1, LHCGR, STAT5B, ITGA2, STAT3, SLC2A8, PRKAR2B, GRB10, 1.70EϪ05/0.030413 stimulus (GO: 0032870) DUSP1, SORBS1, JAK2, GNG2, HDAC9, IGFBP2, INSR, PIK3R1 stimulus. These changes in basal transcription would been observed previously for the estrogen receptor (39– have ripple effects in stromal cells when exposed to a 41) and PGR in uterine fibroids and breast tissue (42, 43). decidual stimulus. In this regard, the PGR transcrip- Distal binding sites have been proposed to function as tome can be classified into 2 distinct categories that enhancers where interaction with promoter regions occurs clearly define the roles of PGR in decidual and nonde- through looping events (42–46). In addition, it has been cidual contexts. suggested that weak binding events not associated with PGR was bound in proximity to nearly 3500 distinct transcriptional changes may require cooperation with co- genes during decidualization. However, genomic enrich- regulators available only in a context- and cell-specific ment analysis revealed that these binding sites were not manner (47). enriched in specific genomic locations or in promoter re- Among the 695 genes differentially regulated with both gions. This nonpromoter enriched binding pattern has a decidual stimulus and PGR knockdown and bound by doi: 10.1210/en.2014-1566 endo.endojournals.org 2249 Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021

Figure 4. Validation of FOSL2 and JUN as direct targets of PGR in HESC decidualization. A, Genome Browser view of PGR binding intervals in proximity to FOSL2 and JUN. B, Validation of PGR binding intervals in proximity to FOSL2 (interval numbers 3566 and 3567) and JUN (176) by ChIP-qPCR analysis. Data represent percent input DNA by qPCR when an antibody against PGR is used for immunoprecipitation and as fold change increase of PGR relative to IgG. Two untranslated regions (utr) are used as negative controls.

PGR are genes known to be direct targets of PGR in human reporter. However, those assays do not account for the endometrial cell and in the murine endometrium, includ- cell-specific expression of binding partners, the dynamic ing FKBP5, HAND2, CYR61, ETS translocation variant interactions of transcription factors with DNA, or the epi- 1(ETV1), collagen ␣-1 (XV) chain (COL15A1), vascular genetic state and accessibility of chromatin to these fac- endothelial growth factor receptor 1 (FLT1), and mono- tors. In this regard, the ChIP-seq profile presented in this amine oxidase A (MAOA) (22, 48). PGR has been shown study is the first comprehensive and unbiased strategy to to bind and activate the promoters of IGFBP1 and PRL in define the global PGR binding landscape in HESC luciferase reporter assays (31, 49). Here we demonstrated decidualization. that in decidualizing HESCs both genes exhibited PGR- PGR-binding regions in decidualizing HESCs con- and FOSL2-dependent expression. FOSL2 and PGR have tained highly enriched motifs for the nuclear hormone re- overlapping ChIP-seq binding intervals 25 kb down- ceptor family. This evidence supports the classic under- stream of PRL, where we hypothesize they may engage in standing of ligand-bound PGR interacting directly with transcriptional cooperation. It has been proposed that the PRE in promoter regions of target genes (51). Whereas PGR-dependent regulation of IGFBP1 occurs via proges- several members of the nuclear receptor family have highly terone induction of FOXO1 and that FOXO1 itself di- similar binding motifs, the enrichment of nuclear receptor rectly binds and regulates IGFBP1 expression in endome- motifs other than the classic PRE suggests a potential co- trial epithelial cells (50). FOXO1 was recently shown to localization of PGR with other nuclear receptors. PGR, bind the promoter region of IGFBP1 and regulate its ex- however, is also known to interact with DNA indirectly, pression in HESC decidualization (28). However, we did associating other transcription factors to exert its effect not observe occupancy of PGR on FOXO1. Instead, we and even without ligand through membrane tyrosine ki- obtained evidence for PGR binding and regulation of nases (52). The enrichment of non-nuclear receptor motifs FOSL2. ChIP-seq analysis showed that FOSL2 occupied in PGR-binding regions included motifs for the forkhead the promoter region of IGFBP1, near the validated domain family, homeobox domain family, and interferon FOXO1 interval. Collectively, this evidence allows us to regulatory factors. FOXO1 and HOXA10 are 2 of the establish a HESC-specific mechanism for the hierarchy in most notable members from each of these families, as they the signaling cascade leading to the regulation of IGFBP1. have been shown to play critical and highly conserved We demonstrated that the global genomic binding pattern roles in mediating PGR action during endometrial stromal of PGR in the decidual context is distinct from that pro- cell decidualization (35, 53, 54). posed by and assessed in cell culture systems. The presence Among the 5 most common binding site motifs en- of PRE in the promoter fragments used in those assays may riched in PGR-binding intervals is the consensus sequence facilitate PGR binding, resulting in the activation of the for the AP-1 family of transcription factors. FOSL2 and 2250 Mazur et al Decidual PGR Transcriptome and Cistrome Endocrinology, June 2015, 156(6):2239–2253 Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021

Figure 5. Requirement of FOSL2 and JUN for the in vitro decidualization of HESC. A, Co-IP of FOSL2 and JUN in decidualized HESCs. B, Proliferation assay of siNT, siFOSL2, siJUN, or siFϩJ transfected cells at day 0 (D0) and 3 (D3) of nondecidual or EPC (decidual) treatments. Error bars represent the SEM from replicates. C, CellMask blue nuclear/cytoplasmic staining of cells treated with siNT, siFOSL2, siJUN, or siFϩJ. D, Gene expression validation by RT- qPCR of FOSL2, JUN, IGFBP1, and PRL in HESCs transfected with siNT or targeting siRNA. Expression data for each gene were normalized to those for 18S rRNA. Data are based on 3 independent experiments. Error bars represent SEM. *, P Ͻ .05, **, P Ͻ .01, n.s., not significant.

JUN were the only AP-1 family members determined to be and FOSL2 intervals exhibited a high degree of overlap. direct targets of PGR, and both were induced in a PGR- This evidence, along with the robust enrichment of AP-1 dependent manner in decidualization. Interestingly, PGR binding elements within PGR intervals, suggests possible doi: 10.1210/en.2014-1566 endo.endojournals.org 2251 Downloaded from https://academic.oup.com/endo/article/156/6/2239/2423101 by guest on 28 September 2021

Figure 6. Shared FOSL2 and PR cistrome in decidualized HESCs. A, Genes bound (within Ϯ10 kb of gene boundaries) by FOSL2 and PGR during HESC decidualization. B, Genome Browser view of FOSL2 and PGR ChIP-seq intervals on 2 shared target genes (TGFBR3 and FKBP5). a PGR-FOSL2 transcriptional cooperation via direct in- and differentiation in keratinocytes (55) and trigger or teraction on chromatin during decidualization. inhibit apoptosis in erythroid cells (56). Significant blunting of the induction of IGFBP1 and In this study, we describe genomic transcription and PRL was observed after FOSL2 and JUN knockdown. PGR-binding in HESCs exposed to an in vitro decidual- Ablation of FOSL2 did not affect the normal pattern of ization stimulus in addition to the transcriptome of de- JUN expression, and, likewise, silencing of JUN did not cidualizing HESCs in which PGR has been silenced. We alter FOSL2 expression. Combined knockdown of both have identified 695 genes likely to be directly regulated by genes resulted in a effect on IGFBP1 and PRL expression PGR during decidualization. Among these targets are the similar to that for the individual knockdowns, perhaps AP-1 transcription factor members FOSL2 and JUN, suggesting that if these 2 factors are cooperating in mediating PGR action in HESC decidualization, knockdown of whose expression was also shown to be required for HESC 1 factor is sufficient to convey the maximum effect in the decidualization. To our knowledge, this is the first effort downstream regulatory pathways. HESCs transfected to characterize the genomic-wide binding profile of these with FOSL2 or JUN, alone or in combination, exhibited a transcription factors and integration with robust expres- highly flatted morphology, indicative of an absent decid- sion profiling techniques to elucidate common targets in ual response and consistent with the significantly attenu- the decidualizing stroma. Most notably, the significant ated expression of the classic markers IGFBP1 and PRL. overlap of PGR-bound and FOSL2-bound genes suggests Interestingly, the cell viability and proliferation assay de- cooperation between these factors in the regulation of termined that only FOSL2 knockdown induced a modest HESC decidualization. increase in proliferation compared with that of all other groups, but only in the standard growth conditions. We observed a decrease in proliferation of HESCs transfected Acknowledgments with siJUN and siFϩJ in the EPC treatment group. The potential role of JUN as a regulator of HESC proliferation We thank Active Motif for performing the ChIP-seq and the is consistent with the observation that changes in the JUN- Integrated Microscopy Core at Baylor College of Medicine for to-JUNB ratios have been shown to regulate proliferation performing HESC imaging. 2252 Mazur et al Decidual PGR Transcriptome and Cistrome Endocrinology, June 2015, 156(6):2239–2253

Address all correspondence and requests for reprints to: decidua-specific enhancer on the human prolactin gene with two Professor Francesco J. DeMayo, Department of Molecular and critical activator protein 1 (AP-1) binding sites. Mol Endocrinol. Cellular Biology, Baylor College of Medicine, One Baylor Plaza, 2001;15:638–653. 15. Tseng L, Tang M, Wang Z, Mazella J. Progesterone receptor (hPR) Mail Stop: BCM130, Houston, TX 77030. E-mail: fdemayo@ upregulates the fibronectin promoter activity in human decidual bcm.edu. fibroblasts. DNA Cell Biol. 2003;22:633–640. This work was supported by the Department of Obstetrics 16. Bamberger AM, Bamberger CM, Gellersen B, Schulte HM. Mod- and Gynecology at Baylor College of Medicine (to E.C.M.) and ulation of AP-1 activity by the human progesterone receptor in en- funding from the National Institutes of Health (Grants R01 dometrial adenocarcinoma cells. Proc Natl Acad Sci USA. 1996;93: 6169–6174. HD042311 and U54 HD007495 to F.J.D.) 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