Abstract
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Progesterone Receptor Transcriptome and Cistrome in Decidualized Human Endometrial Stromal Cells
Abstract
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 17β-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.
The endometrium is a dynamic tissue regulated by the ovarian steroid hormones, estradiol and progesterone. The endometrium proliferates under the influence of estradiol produced by growing follicles during the first 2 weeks of the human menstrual cycle. After ovulation, progesterone from the newly formed corpus luteum drives a process of differentiation during which the endometrium becomes competent to receive and support the growth of the embryo (1). The hallmark of this differentiation or decidualization, occurs in the stromal compartment of the endometrium where spindle-shaped fibroblasts transform to plump secretory cells and provide a histiotrophic environment that nourishes the developing embryo while at the same time limiting the invasiveness of the trophoblast, a supportive task required for placentation. The decidua also creates an interphase where the maternal immunity is modulated to tolerate the fetal allograft (2). Because this process is critical to establishing and maintaining pregnancy, it is likely that defective decidualization underlies a certain proportion of infertility in women (3, 4).
The fundamental regulator of endometrial stromal cell decidualization is progesterone, which acts through its nuclear steroid hormone receptor, the progesterone receptor (PGR) (5). Classically, nuclear steroid hormone receptors bind DNA directly at specific hormone response elements in promoter regions and drive the transcription or repression of particular genes (6). These receptors can also interact with DNA indirectly through protein-protein interactions with other factors that, in turn, bind DNA. Regardless of direct or indirect DNA binding, nuclear steroid hormone receptors associate with complexes of coregulators that are responsible for the events required to drive or repress transcription (7). Steroid hormones are known to regulate a variety of seemingly opposing processes, including proliferation and differentiation and, consequently have different effects in different tissues. These context-specific actions are defined by the intracellular milieu and the intrinsic expression of different components of these receptor-coregulator complexes (8). Identification of relevant PGR coregulators and downstream signaling effectors is critical to the understanding of the endometrial stromal cell–specific transcriptional changes that occur in the decidualization process.
It has been shown that the activator protein-1 (AP-1) family of transcription factors are involved in the regulation of gene expression during cell differentiation and that there is extensive cross talk between AP-1 and nuclear receptors (9,–11). AP-1 is composed of homodimers or heterodimers of members of the Fos and Jun families (FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND), and the different combinations of these members have been described in various contexts in which AP-1 is thought to act. The AP-1 member Jun dimerization protein 2 (JDP-2) has been shown to interact directly with the transcription activation function (AF) domain of PGR and increase hormone-dependent PGR-mediated transactivation primarily by stimulating AF-1 activity (12). FOSL1 has been described as a downstream effector of the phosphatidylinositol 3-kinase/AKT signaling pathway responsible for development of trophoblast lineages integral to establishing the maternal-fetal interface, highlighting the importance of AP-1 factors in establishment of pregnancy (13). The expression of prolactin (PRL), a major secretory product of decidualized endometrium, has been shown to be regulated via 2 AP-1 sites in the gene promoter sites, which bind specific members of the AP-1 family and not others (14). Furthermore, it was shown that PGR regulates the promoter activity of fibronectin 1 (FN1), a critical component of the extracellular matrix involved in cell adhesion and migration, mainly through the CRE/AP-1 site located in the proximal region of the promoter in human decidual fibroblasts (15). Last, progesterone has been shown to regulate AP-1 activity in human endometrial adenocarcinoma cells and has been explained as a mechanism by which progesterone inhibits endometrial cancer cell growth (16, 17).
The aim of this study was to increase our understanding of the transcriptional changes that occur during the in vitro decidualization of primary human endometrial stromal cells (HESCs) by taking advantage of recent advances in next-generation sequencing technologies. RNA sequencing (RNA-seq) offers an important improvement over microarrays because of the capture of a higher dynamic range of expression levels that more faithfully describe the robust changes occurring during the secretory transformation of stromal cells. Second, we aimed to determine the role of PGRs in mediating the expression changes during decidualization by describing the transcriptome in PGR-silenced cells exposed to a decidual stimulus. Third, we aimed to explore the direct role of PGRs in this process by describing the whole-genome binding profile of PGR during decidualization using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). With the integration of these robust data sets, we identified key factors whose expression changes during decidualization in a PGR-dependent manner, among them the AP-1 family member FOSL2. Finally, we determined that a vast majority of PGR-bound genes are also bound by FOSL2 and propose that this overlap may underlie a mechanism of transcriptional cooperation via direct interaction on chromatin during decidualization. The data generated by this study are important to better understand the direct and indirect mechanisms underlying PGR action and the involvement of the AP-1 family of transcription factors in the endometrium.
Materials and Methods
Endometrial stromal cells
Human endometrial samples were obtained from 6 healthy, reproductive-aged volunteers with regular menstrual cycles and no history of gynecological malignancies under a human subject protocol approved by the institutional review board of Baylor College of Medicine. After receipt of informed consent from participants, an endometrial biopsy was performed during the follicular phase of the menstrual cycle, and endometrial stromal cells were isolated by enzymatic digestion and filtration, as described previously (4, 18). Stromal cells were cultured in DMEM/F12 media with 10% fetal bovine serum, and all experiments were carried out within 4 cell passages. When cells reached approximately 60% confluence, they were transfected with 60 nM scrambled, nontargeting (siNT) or PGR-targeting (siPGR) small interfering RNA (siRNA) (ON-TARGETplus; GE Dharmacon, Lafayette, CO) using Lipofectamine RNAiMAX lipid (Life Technologies) per the manufacturer's instructions. After 48 hours of transfection exposure, cells were either collected or exposed to 10 nM 17β-estradiol, 100 nM medroxyprogesterone acetate, and 1 mM 8-bromo-cAMP (EPC) in Opti-MEM I (Life Technologies) reduced serum media supplemented with 2% stripped fetal bovine serum, antibiotics, and antimycotics for 72 hours. For quantitative PCR experiments, mRNA was isolated by TRIzol (Life Technologies) extraction per the manufacturer's protocol. For cell viability assays, 103 HESCs were plated per well (96-well plate) 48 hours after transfection with siRNA. After adherence to the well floor, cells were grown in DMEM/F12 media with 10% fetal bovine serum (nondecidual) or EPC media (decidual). Cell viability was determined using the CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega) according to the manufacturer's instructions.
RNA-seq
RNA was purified from HESC cultures established from 3 independent biopsy samples and treated as described above for RNA-seq analysis using the Ambion RiboPure Kit (Life Technologies). RNA-seq was performed for each individual patient sample. Raw reads were mapped to human genome hg19 and splice junction sites using Bowtie (v0.12.7) (19) and TopHat (v2.0.0) (20) with the strand-specific model that matches our dUTP library construction protocol. The human annotation 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/overview.html) using the default model. Differential gene expression was analyzed with R (v2.14.0; http://www.R-project.org) and the Bioconductor edgeR package (edgeR_2.4.6) (21). With edgeR, we fit a negative binomial generalized log-linear model to the read counts for each gene by accounting for both patient and treatment in the design. Gene-wise statistical tests were conducted to identify genes that have consistent changes in response to treatment across 3 individuals. A false discovery rate (FDR) of 0.05 was used as the cutoff for significant differential expression.
Gene expression changes observed by RNA-seq were validated in replicate experiments by real-time RT-quantitative PCR(qPCR). mRNA was isolated by TRIzol (Life Technologies) extraction and reverse transcribed into cDNA with Moloney murine leukemia virus (Life Technologies) per the manufacturer's protocol. Expression levels of mRNA were determined by qPCR on a QuantStudio 12K Flex Real-Time qPCR system (Life Technologies) using iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) and primers (Sigma-Aldrich) and normalized to 18s RNA (Supplemental Table 1). One-way ANOVA followed by a Tukey-Kramer multiple comparisons test was used to compare gene expression levels.
ChIP-seq
PGR, FOSL2, and input ChIP were performed by Active Motif, Inc on HESC cultures established from the 6 endometrial biopsy specimens as described previously (18, 22). The model-based analysis of ChIP-seq (23) algorithm was used to find peaks by normalizing PGR and FOSL2 ChIP against the input control with a cutoff of P < 10−10. Associated genes were called if PGR or FOSL2 intervals were located within ±10 kb of the gene boundaries. Analyses of gene distribution and enriched motifs were performed using Cistrome (http://cistrome.org/ap/) (24). Gene functional analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov/) (25, 26). The PGR ChIP-seq data set was also used in 2 additional publications, one in which a comparative analysis of ancient mammalian transposable elements involved in decidualization was performed and the second one in which the requirement of forkhead box protein O1 (FOXO1) for PGR binding in decidualization was evaluated (27, 28).
ChIP-seq validation
ChIP-seq PGR intervals on FOSL2 and JUN were confirmed by ChIP-qPCR. HESCs were grown to 80% confluence before exposure to a decidual stimulus (EPC). After 72 hours of exposure, ChIP was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) following the manufacturer's instructions. In brief, proteins were cross-linked to DNA with formaldehyde, and the cells were treated with micrococcal nuclease and mechanical shearing to digest DNA and break nuclear membranes. This sheared chromatin was immunoprecipitated with antibodies to PGR and IgG (H-190/sc-7208 and sc-2027, Santa Cruz Biotechnology, Inc) (Table 1). Primers for SYBR Green RT-qPCR were designed to query the PGR intervals on FOSL2 and JUN as determined by the ChIP-seq (Supplemental Table 1). Two untranslated regions were queried as a negative control (Human ChIP Control qPCR Primer Set, Active Motif). qPCR was used to compare PGR-immunoprecipitated to IgG-precipitated chromatin. The presence of putative hormone response elements for PGR within the FOSL2 and JUN intervals was determined using the transcription factor binding profile database, JASPAR (http://jaspar.genereg.net/).
Table 1.
Antibody Table
| Peptide/Protein Target | Antigen Sequence (If Known) | Name of Antibody | Manufacturer, Catalog No, and/or Name of Individual Providing the Antibody | Species Raised in (Monoclonal or Polyclonal) | Dilution Used |
|---|---|---|---|---|---|
| PGR | Amino acids 375–564 of PGR of human origin | PR (H-190) | Santa Cruz, sc-7208 | Rabbit polyclonal | 4 μg/immunoprecipitation reaction |
| Fra-2 (FOSL2) | Epitope mapping at the N terminus of Fra-2 of human origin | Fra2 (Q-20) | Santa Cruz, sc-604 | Rabbit polyclonal | 4 μg/immunoprecipitation reaction |
| Normal rabbit IgG | Normal rabbit IgG | Santa Cruz, sc-2027 | Rabbit | 4 μg/immunoprecipitation reaction |
Analysis of AP-1 factors
qPCR was used to investigate the effect of silencing FOSL2 and JUN in HESCs exposed to a decidual stimulus. As described above, HESCs were transfected with siRNA targeting FOSL2, JUN, or a dual knockdown of FOSL2 and JUN. Transfected cells 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 SYBR Green protocol and primers designed using the Primer-BLAST suite at the National Center for Biotechnology Information (NCBI) and normalized to 18s RNA (Supplemental Table 1).
Coimmunoprecipitation (Co-IP) of FOSL2 and JUN
Co-IP of FOSL2 and JUN was performed using the Universal Magnetic Co-IP kit (catalog no. 54002; Active Motif) as per the manufacturer's instructions. In brief, HESCs were grown to 70% confluence and treated with EPC for 72 hours to stimulate decidualization as described above. One hour before harvest, cells were treated with fresh media and hormones. Cell collection, whole-cell extraction, and quantification were performed as per the manufacturer's instructions. Co-IP was performed with 400 μg of protein and 1 μg of IgG, FOSL2, and JUN antibodies (catalog nos. sc-2027, sc-604, and sc-1694; Santa Cruz Biotechnology) for 3 hours. Magnetic beads were incubated with the antibody/extract mixtures (1 hour, 4°C) and subsequently washed 4 times with complete/Co-IP wash buffer. Bead pellets were resuspended in 2× reducing loading buffer (catalog no. 161–0737; Bio-Rad Laboratories) containing 10% β-mercaptoethanol and boiled for 5 minutes at 90°C before electrophoresis separation in Bis-Tris NuPAGE 4%–12% gels (1.0 mm, 10-well; catalog no. NP0321BOX, Novex; Life Technologies). Proteins were transferred to polyvinylidene difluoride membranes (EMD Millipore) in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) (Life Technologies). polyvinylidene difluoride membranes were subsequently blocked with 5% blotting grade nonfat milk (catalog no. 170–6404; Bio-Rad) in PBS containing 0.1% Tween 20 (PBST) for 1 hour at room temperature. Membranes were probed with antibodies for FOSL2 and JUN overnight at 4°C in 5% blotting grade nonfat milk in PBST. Blotted membranes were subsequently washed 3 times with PBST and incubated for 1 hour (room temperature) with anti-rabbit peroxidase secondary antibody. Blots were washed 3 times with PBST and subsequently 3 times with PBS. Luminol-based detection of bands on film was performed using the Amersham ECL Western Blotting System (GE Healthcare) as per the manufacturer's instructions.
Imaging of HESC morphology
HESCs were grown on coverslips and treated with EPC for 3 days to stimulate a decidual response as described above. Cells were washed with cold PBS once and fixed with 4% formaldehyde in PEM buffer (potassium PIPES [pH 6.8], 5 mM EGTA [pH 7.0], and 2 mM MgCl2) for 30 minutes. Fixative was removed, and cells were washed 3 times with PEM alone. Cell delineation was determined by staining with HCS CellMask Blue (catalog no. H32720; Life Technologies) diluted 1:100 000 in 0.1% Triton X-100 in PEM for 15 minutes and subsequently washed 3 times with PEM. Coverslips were mounted on slides with SlowFade Gold (catalog no. S-2828; Molecular Probes). Images were acquired using a GE Healthcare DeltaVision Image Restoration microscope with a 40x/0.95na objective. Optical sections (0.35-mm; z-stacks) were taken and deconvolved using softWoRx software, and maximum intensity was projected.
Results
Transcriptome of decidualizing HESCs
To identify the mRNA profile change during HESC decidualization, RNA-seq was performed in HESCs transfected with siNT with and without a hormone stimulus. A total of 4061 genes were differentially regulated between nondecidual and decidual HESCs (Supplemental Table 2). Table 2 summarizes the top pathways enriched in the functional pathway analysis performed with DAVID bioinformatics resources (25, 26). Enrichment was seen in pathways for cell communication, cell cycle, and metabolism, consistent with the expected processes in cells undergoing decidualization. Decidualizing HESCs are characterized by cell enlargement, nuclear rounding, expansion of the Golgi/endoplasmic reticulum, and accumulation of glycogen and lipid in the cytoplasm. These cells are active secretory cells with secreted products commonly used as markers of decidualization. Most commonly, IGF binding protein-1 (IGFBP-1) and PRL have been monitored as molecular markers for the decidualization process, and in our data, IGFBP1 and PRL are 2 of the most highly induced genes with exposure to EPC. These data confirmed the induction of cytokines (IL11), growth factors (heparin-binding epidermal growth factor [HBEGF]), neuropeptides (somatostatin), and critical transcription factors (FOXO1), which are expressed in decidualizing stromal cells at high levels and thought to amplify and propagate the decidual process (1). Furthermore, decidualizing HESCs contain surface projections that extend both into the extracellular matrix and into adjacent cells, and adherens junctions are found between adjacent cells. Data confirmed expression changes in decorin, laminins, type IV collagen, and fibronectin, which are known to be involved in the remodeling of decidual cells. Among the signaling pathways enriched were TGF-β, Janus kinase (Jak)-signal transducer and activator of transcription (STAT), and wingless-related integration site (Wnt) pathways.
Table 2.
DAVID Functional Analysis Using KEGG Pathways for Genes With Differential Expression (FDR < 0.05) by RNA-Seq in HESCs Before and After Treatment With a Decidual Stimulus (EPC)
| Description, (KEGG hsa Identification No.) | Genes |
|---|---|
| Cell communication | |
| 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, COL5A1, COL4A6, LAMA2, LAMA1, ITGA9, ITGA6, FYN, ITGA8, ITGA7, MYLK | |
| FGF5, ENAH, PDGFB, FGF9, BCAR1, IQGAP3, IQGAP2, INSRR, VCL, ACTG1, GSN, 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 | |
| B4GALT1, ST6GAL1, GANAB, MAN1A2, FUT8, ALG1, ALG2, ALG3, ALG5, MOGS, ALG9, LOC151162, STT3A, RPN1, DPM3, RPN2, MGAT5, DDOST | |
| SAT1, ODC1, ALDH18A1, ASS1, MAOA, MAOB, ALDH3A2, CKB, GLUL, PYCR2, P4HA2, P4HA1, GLS, P4HA3, ALDH2, OAT, SMS | |
| ARSB, SGSH, HGSNAT, GNS, NAGLU, HYAL3, HPSE, GUSB, GLB1 | |
| 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 (4914) | ADCY3, CDK1, ADCY4, PIK3CD, IGF1, PDE3B, PKMYT1, IGF2, MAPK10, CDC25C, PPP1CB, CDK2, PRKX, CDC25B, CCNB1, RPS6KA6, CCNB2, MAD2L1, PLK1, BUB1, MAPK8, CCNA2, PIK3R1, AKT3 |
| Signal transduction | |
| BMP4, PPP2R1B, NOG, LTBP1, SMAD7, RBL1, BMPR2, TGFB3, SMAD3, DCN, SMAD1, TGFB2, INHBA, CDKN2B, ID2, INHBE, ID1, LEFTY2, ID4, SMURF2, ID3, THBS1, THBS2, MYC | |
| 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 | |
| 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 |
To evaluate the role of PGR in the regulation of decidual genes, HESCs were transfected with siRNA targeting PGR before decidual stimulus (siPGR EPC). A total of 4960 genes were differentially regulated with PGR knockdown compared with those for siNT EPC (Supplemental Table 2). Of the genes, 52% that were differentially regulated during decidualization were also regulated when siPGR EPC cells were compared with siNT EPC cells. Pathway analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) functional groups revealed distinct biological pathways for the 3 resulting gene groups: genes changing with decidual stimulus (siNT vehicle vs siNT EPC), genes whose expression changed only with siPGR transfection (siNT EPC vs siPGR EPC), and genes with expression changes both during decidualization (siNT vehicle vs siNT EPC) and with PGR knockdown (siNT EPC vs siPGR EPC). Distinct biological processes were evident in genes differentially expressed with decidualization, genes with expression changes with PGR knockdown, and genes both differentially regulated with decidualization and PGR knockdown. Results from the DAVID analysis suggest that the TGF-β pathway and cell cycle regulation are differentially regulated with decidualization alone, metabolic pathways are enriched with PGR knockdown alone, and adhesion, cytoskeleton, Jak-STAT, and Wnt pathways are both PGR-regulated and enriched with decidualization (Figure 1A). In the analysis of the PGR transcriptome, genes fall in 2 distinct categories with very clear decidual and nondecidual biology. The EPC-regulated and siPGR-regulated genes enrich for Gene Ontology (GO) terms including regulation of proliferation, vascular development, apoptosis, migration, adhesion, and cytoskeletal organization (Supplemental Table 3). These processes are hallmarks of the robust transformation stromal fibroblasts undergo during decidualization. In contrast, the siPGR-regulated genes that are not regulated with EPC decidualization enrich for terms related to protein modification, catabolism, and localization (Supplemental Table 4).
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 < .05, **, P < .01, n.s., not significant.
Representative examples of qPCR validation of gene expression changes detected by RNA-seq are shown in Figure 1B. Frizzled-10 (FZD10) is induced with exposure to EPC (siNT + EPC) but is not dependent on PGR as siPGR does not change the gene induction with decidualization (siPGR + EPC). Similarly, the gene encoding the Krüppel-like transcription factor GLIS family zinc finger 2 (GLIS2) is down-regulated with decidualization in a PGR-independent fashion. Signal transducer and activator of transcription 3 (STAT3) and Dikkopf-related protein 1 (DKK1) are PGR dependent. Induction of STAT3 and DKK1 with decidualization (siNT + EPC) is blunted with knockdown of PGR (siPGR + EPC). Certain genes (Mothers against decapentaplegic homolog 4 [SMAD4] and the transcription factor specificity protein 1 [SP1]) do not exhibit changes in expression with decidualization (siNT + EPC) but are down-regulated upon silencing of PGR (siPGR + EPC).
Identification of PGR global genomic binding sites in HESCs using ChIP-seq
To identify direct targets of PGR in HESC decidualization, PGR association with DNA was assessed by ChIP-seq. The binding of PGR was defined to 8690 intervals. and 5091 of these intervals were located within 10 kb of gene boundaries of 3478 genes (Supplemental Table 3). CEAS enrichment analysis showed no significant enrichments for specific genomic boundaries relative to those for the genome reference (Figure 2A) or the promoter region of genes (Figure 2B). Motif analysis of PGR intervals using the SeqPos tool in the Cistrome data analysis platform (24) revealed enrichment in binding sequences for the steroid nuclear hormone receptors (including PGR), forkhead domain, homeobox, interferon regulatory factor, and leucine zipper factors (Figure 2C). The nuclear hormone response element is the most highly enriched binding motif in the PGR cistrome, suggesting that PGR is frequently bound to the progesterone response element (PRE) during HESC decidualization and near regions recognized by factors such as FOXO1, HOXA11, IRF4, and AP-1 family members.
ChIP-seq analysis of PGR cistrome. A, CEAS enrichment analysis of PGR intervals 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 analyzed with the SeqPos tool in Cistrome. The 5 most highly enriched consensus binding motifs in PGR intervals by Cistrome analysis, P < 10−30. UTR, untranslated region.
Overall, 695 genes were found to be regulated during decidualization, to be differentially regulated with PGR knockdown, and to contain bound PGR during decidualization (Figure 3A). DAVID analysis revealed that the direct PGR target genes enriched biological themes for regulation of cell motion, vascular development, and intracellular cascade (Table 3). Among the known direct PGR target genes involved in decidualization, we observed PGR binding and differential regulation in several genes such as FK506 binding protein 5 (FKBP5), heart- and neural crest derivatives-expressed protein 2 (HAND2), and cysteine-rich angiogenic inducer 61 (CYR61) (29, 30). ChIP-seq did not identify binding of PGR to the promoter of IGFBP1, a finding that was inconsistent with previous reports that demonstrated progesterone-responsive regulation of promoter activity (31). IGFBP1 expression was significantly attenuated with PGR silencing in decidualizing HESCs. Similarly, binding of PGR was not observed proximal to the PRL gene but instead was located 25 kb downstream. Consistent with previous reports, we observed differential regulation of FGF9, WNT2, CNR1, ARNT2, and GATA6 with siPGR (5). However, only ARNT2 was bound by PGR within 10 kb of official gene annotations. Other known progesterone-regulated targets such as the ZEB1, KLF15, HOXA10, and PTGS1 were also found to have PGR binding sites and undergo differential regulation with siPGR (Supplemental Table 2) (32,–36).
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 regulated in HESCs transfected with siRNA targeting PGR before EPC treatment. B, Gene expression validation of PGR knockdown and direct gene targets by RT-qPCR and normalization with 18s rRNA. Data are based on 3 independent experiments. Error bars represent SEM. *, P < .05. D0, day 0; D3, day 3.
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: 0051270) | DLC1, RTN4, IL6ST, STAT5B, ITGB3, TPM1, VCL, TRIB1, NISCH, S1PR1, TEK, SCARB1, THBS1, INSR, PIK3R1, COL18A1, IRS2, ACTN4, PDPN, ITGA2, PTPRU, LAMA2, CDH13, LAMA1, LAMA4, ETS1, F3, RRAS2, PDGFRA, TGFBR3, JAK2, HDAC9, F2R | 3.62E−12/6.47E−09 |
| Vasculature development (GO: 0001944) | RTN4, ZFAND5, COL3A1, TIPARP, ENPEP, ELK3, CDH2, WT1, SEMA5A, SHB, APOB, 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 | 1.28E−11/2.29E−08 |
| Intracellular signaling cascade (GO: 0007242) | ADCY3, IL6ST, LHCGR, STAT5B, TNFSF15, IQGAP2, TLR4, LPAR1, RGL1, PRKAR2B, 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 | 9.26E−09/1.65E−05 |
| Cell adhesion (GO: 0007155) | DLC1, CLSTN2, TLN2, CLSTN1, LMO7, EDIL3, VCL, ARHGAP6, S1PR1, WISP1, 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 | 4.72E−08/8.43E−05 |
| Enzyme-linked receptor protein signaling pathway (GO: 0007167) | ZFAND5, LTBP2, IL6ST, COL3A1, TIPARP, STAT5B, GREM2, SLC2A8, EPHB6, SORBS1, CTGF, TEK, PDGFC, SHC3, NRG1, FGF1, FRS2, INSR, PIK3R1, PTPRJ, PLAT, IRS2, NIN, MYO1E, PTPRU, STAT3, GRB10, ID1, JUN, PDGFRA, ROR1, TGFBR3, SORT1, ROR2, JAK2, ENG | 1.98E−07/3.53E−04 |
| Response to organic substance (GO: 0010033) | ADCY3, ATP6V0E1, IL6ST, LHCGR, ARNT2, STAT5B, SNCA, TLR4, SLC2A8, 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 | 1.52E−06/0.002708 |
| Extracellular structure organization (GO: 0043062) | RECK, COL18A1, MYO6, TNC, ADAMTSL4, ELN, COL3A1, MAP1B, NID1, DCN, CDH2, CACNB4, COL5A2, COL5A1, COL14A1, CRISPLD2, PDGFRA, COL11A1, ENG, F2R, CYR61 | 5.89E−06/0.01051 |
| Protein kinase cascade (GO: 0007243) | IL6ST, STAT5B, DUSP10, TNFSF15, NFKBIA, TLR4, MAPKAPK2, LPAR1, TRIB1, 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 | 9.09E–06/0.01622 |
| Response to wounding (GO: 0009611) | TPST1, MASP1, TNC, STAT5B, COL3A1, AFAP1L2, TLR4, C1R, ITGB3, C1S, ELK3, 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 | 1.09E−05/0.019512 |
| Cellular response to hormone stimulus (GO: 0032870) | ADCY3, IRS2, ENPP1, LHCGR, STAT5B, ITGA2, STAT3, SLC2A8, PRKAR2B, GRB10, DUSP1, SORBS1, JAK2, GNG2, HDAC9, IGFBP2, INSR, PIK3R1 | 1.70E−05/0.030413 |
Role of AP-1 in HESC decidualization
The genes differentially regulated with decidualization and PGR silencing while containing bound PGR during decidualization included 2 members of the AP-1 family of transcription factors. Fos-like antigen 2 (FOSL2 or FRA2) and jun proto-oncogene (JUN) were each up-regulated with exposure to a decidual stimulus and down-regulated 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 (Supplemental Figure 1). This analysis revealed that whereas FOSL1 undergoes hormone dependent down-regulation during decidualization, other members of the AP-1 family are not differentially regulated. Furthermore, the expression of these members is unaffected 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, immediately upstream and approximately 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 (Supplemental Table 5).
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.
The association of FOSL2 and JUN during decidualization was evaluated by performing Co-IP in decidualized HESCs. Immunoprecipitation 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 evaluation of the role of FOSL2 and JUN in proliferation and decidualization. HESCs were transfected with scrambled siRNA (siNT) and FOSL2-targeting siRNA (siFOSL2), JUN-targeting siRNA (siJUN), and FOSL2 + JUN-targeting siRNA (siF+J) and cultured in standard growth media and EPC media. At day 0, the cell proliferation and viability assay revealed no differences between the groups for either treatment. At day 3, we observed an increase in the proliferation of cells transfected with siFOSL2 only in the nondecidual conditions. In addition, we observed a decrease in proliferation in the siJUN and siF+J groups under decidualizing conditions (Figure 5B).
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 siF+J 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 siF+J. 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 < .05, **, P < .01, n.s., not significant.
After 6 days of hormone stimulus, HESCs transfected with siNT displayed a decidual, rounded morphology. HESCs transfected with siFOSL2, siJUN, and siF+J displayed highly flattened and elongated fibroblastic cell morphology indicative of an absent decidual transformation (Figure 5C). qPCR analysis confirmed a lack of FOSL2 and JUN induction when cells were transfected with targeting siRNA. In addition, inductions of IGFBP1 and PRL were blunted by knockdown of FOSL2 and JUN (Figure 5D).
When genomic DNA binding of FOSL2 was assessed by ChIP-seq in decidualizing HESCs, 24 298 intervals of FOSL2-bound DNA were found, representing proximity (±10 kb) to 8586 distinct genes. FOSL2 was thus found to bind more than twice as many genes as PGR. Furthermore, nearly 80% (2709 of 3477) of PGR-bound genes were likewise bound by FOSL2. In examination of the genes targeted by both PGR and FOSL2, a large degree of interval overlap was observed. Examples are shown in Figure 6, in which ChIP-seq binding intervals are displayed on the UCSC Genome Browser for the TGFBR3 and FKBP5 gene loci.
Shared FOSL2 and PR cistrome in decidualized HESCs. A, Genes bound (within ±10 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).
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 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: vehicle-treated HESCs transfected with nontargeting siRNA and EPC-treated HESCs transfected with PGR-targeting siRNA. One limitation of this strategy is the secondary effects of transfection on gene expression changes that occur during decidualization. However, despite these limitations, the robust power of this RNA-seq strategy was able to provide greater sensitivity than standard expression arrays in which a similar transfection and treatment strategy was used (3).
We subsequently compared the genes regulated in decidualization (siNT vehicle vs siNT EPC) and the PGR-dependent genes (siNT EPC and siPGR EPC) to discern genes that were regulated during decidualization in a PGR-dependent manner. In this comparison, we observed that a subset of PGR-dependent genes do not overlap with the decidual gene set (2830 genes). GO analysis revealed that the PGR-only transcriptional profile constitutes a nondecidual biology. It has been shown that unliganded PGR is able to regulate expression of genes by binding to genomic sites with repressive protein complexes to silence gene expression in T47D breast cancer cells (38). Although expression and transcriptional activity of PGR appear to peak in decidual conditions when hormone is present, it is possible that in nondecidual conditions PGR is engaged in transcriptional repression. Ablation of PGR would consequently result in changes in gene expression in the basal state of stromal fibroblasts before hormone stimulus. These changes in basal transcription would have ripple effects in stromal cells when exposed to a decidual stimulus. In this regard, the PGR transcriptome can be classified into 2 distinct categories that clearly define the roles of PGR in decidual and nondecidual contexts.
PGR was bound in proximity to nearly 3500 distinct genes during decidualization. However, genomic enrichment analysis revealed that these binding sites were not enriched in specific genomic locations or in promoter regions. This nonpromoter enriched binding pattern has been observed previously for the estrogen receptor (39,–41) and PGR in uterine fibroids and breast tissue (42, 43). Distal binding sites have been proposed to function as enhancers where interaction with promoter regions occurs through looping events (42,–46). In addition, it has been suggested that weak binding events not associated with transcriptional changes may require cooperation with coregulators available only in a context- and cell-specific manner (47).
Among the 695 genes differentially regulated with both a decidual stimulus and PGR knockdown and bound by PGR are genes known to be direct targets of PGR in human endometrial cell and in the murine endometrium, including FKBP5, HAND2, CYR61, ETS translocation variant 1 (ETV1), collagen α-1 (XV) chain (COL15A1), vascular endothelial growth factor receptor 1 (FLT1), and monoamine oxidase A (MAOA) (22, 48). PGR has been shown to bind and activate the promoters of IGFBP1 and PRL in luciferase reporter assays (31, 49). Here we demonstrated that in decidualizing HESCs both genes exhibited PGR- and FOSL2-dependent expression. FOSL2 and PGR have overlapping ChIP-seq binding intervals 25 kb downstream of PRL, where we hypothesize they may engage in transcriptional cooperation. It has been proposed that the PGR-dependent regulation of IGFBP1 occurs via progesterone induction of FOXO1 and that FOXO1 itself directly binds and regulates IGFBP1 expression in endometrial epithelial cells (50). FOXO1 was recently shown to bind the promoter region of IGFBP1 and regulate its expression in HESC decidualization (28). However, we did not observe occupancy of PGR on FOXO1. Instead, we obtained evidence for PGR binding and regulation of FOSL2. ChIP-seq analysis showed that FOSL2 occupied the promoter region of IGFBP1, near the validated FOXO1 interval. Collectively, this evidence allows us to establish a HESC-specific mechanism for the hierarchy in the signaling cascade leading to the regulation of IGFBP1. We demonstrated that the global genomic binding pattern of PGR in the decidual context is distinct from that proposed by and assessed in cell culture systems. The presence of PRE in the promoter fragments used in those assays may facilitate PGR binding, resulting in the activation of the reporter. However, those assays do not account for the cell-specific expression of binding partners, the dynamic interactions of transcription factors with DNA, or the epigenetic state and accessibility of chromatin to these factors. In this regard, the ChIP-seq profile presented in this study is the first comprehensive and unbiased strategy to define the global PGR binding landscape in HESC decidualization.
PGR-binding regions in decidualizing HESCs contained highly enriched motifs for the nuclear hormone receptor family. This evidence supports the classic understanding of ligand-bound PGR interacting directly with PRE in promoter regions of target genes (51). Whereas several members of the nuclear receptor family have highly similar binding motifs, the enrichment of nuclear receptor motifs other than the classic PRE suggests a potential colocalization of PGR with other nuclear receptors. PGR, however, is also known to interact with DNA indirectly, associating other transcription factors to exert its effect and even without ligand through membrane tyrosine kinases (52). The enrichment of non-nuclear receptor motifs in PGR-binding regions included motifs for the forkhead domain family, homeobox domain family, and interferon regulatory factors. FOXO1 and HOXA10 are 2 of the most notable members from each of these families, as they have been shown to play critical and highly conserved roles in mediating PGR action during endometrial stromal cell decidualization (35, 53, 54).
Among the 5 most common binding site motifs enriched in PGR-binding intervals is the consensus sequence for the AP-1 family of transcription factors. FOSL2 and JUN were the only AP-1 family members determined to be direct targets of PGR, and both were induced in a PGR-dependent manner in decidualization. Interestingly, PGR and FOSL2 intervals exhibited a high degree of overlap. This evidence, along with the robust enrichment of AP-1 binding elements within PGR intervals, suggests possible a PGR-FOSL2 transcriptional cooperation via direct interaction on chromatin during decidualization.
Significant blunting of the induction of IGFBP1 and PRL was observed after FOSL2 and JUN knockdown. Ablation of FOSL2 did not affect the normal pattern of JUN expression, and, likewise, silencing of JUN did not alter FOSL2 expression. Combined knockdown of both genes resulted in a effect on IGFBP1 and PRL expression similar to that for the individual knockdowns, perhaps suggesting that if these 2 factors are cooperating in mediating PGR action in HESC decidualization, knockdown of 1 factor is sufficient to convey the maximum effect in the downstream regulatory pathways. HESCs transfected with FOSL2 or JUN, alone or in combination, exhibited a highly flatted morphology, indicative of an absent decidual response and consistent with the significantly attenuated expression of the classic markers IGFBP1 and PRL. Interestingly, the cell viability and proliferation assay determined that only FOSL2 knockdown induced a modest 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 with siJUN and siF+J in the EPC treatment group. The potential role of JUN as a regulator of HESC proliferation is consistent with the observation that changes in the JUN-to-JUNB ratios have been shown to regulate proliferation and differentiation in keratinocytes (55) and trigger or inhibit apoptosis in erythroid cells (56).
In this study, we describe genomic transcription and PGR-binding in HESCs exposed to an in vitro decidualization stimulus in addition to the transcriptome of decidualizing HESCs in which PGR has been silenced. We have identified 695 genes likely to be directly regulated by PGR during decidualization. Among these targets are the AP-1 transcription factor members FOSL2 and JUN, whose expression was also shown to be required for HESC decidualization. To our knowledge, this is the first effort to characterize the genomic-wide binding profile of these transcription factors and integration with robust expression profiling techniques to elucidate common targets in the decidualizing stroma. Most notably, the significant overlap of PGR-bound and FOSL2-bound genes suggests cooperation between these factors in the regulation of HESC decidualization.
Acknowledgments
We thank Active Motif for performing the ChIP-seq and the Integrated Microscopy Core at Baylor College of Medicine for performing HESC imaging.
This work was supported by the Department of Obstetrics and Gynecology at Baylor College of Medicine (to E.C.M.) and funding from the National Institutes of Health (Grants R01 HD042311; and U54 HD007495; to F.J.D.) The Integrated Microscopy Core at Baylor College of Medicine was also supported with funding from the National Institutes of Health (Grants HD007495;, DK56338;, and CA125123), the Dan L. Duncan Cancer Center, and the John S. Dunn Gulf Coast Consortium for Chemical Genomics.
Disclosure Summary: The authors have nothing to disclose.
Footnotes
Abbreviations:
- AP-1
- activator protein-1
- ChIP-seq
- chromatin immunoprecipitation followed by deep sequencing
- Co-IP
- coimmunoprecipitation
- DAVID
- Database for Annotation, Visualization, and Integrated Discovery
- EPC
- estradiol, medroxyprogesterone acetate, 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
- siF+J
- 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.
References
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Funding
Funders who supported this work.
NCI NIH HHS (2)
Grant ID: CA125123
Grant ID: P30 CA125123
NICHD NIH HHS (6)
Grant ID: HD007495
Grant ID: R01HD042311
Grant ID: T32 HD007495
Grant ID: P30 HD007495
Grant ID: R01 HD042311
Grant ID: U54 HD007495
NIDDK NIH HHS (2)
Grant ID: P30 DK056338
Grant ID: DK56338