Pioneer Factors FOXA1 and FOXA2 Assist Selective Glucocorticoid Receptor Signaling in Human Endometrial Cells

Abstract

Successful pregnancy relies on dynamic control of cell signaling to achieve uterine receptivity and the necessary biological changes required for endometrial decidualization, embryo implantation, and fetal development. Glucocorticoids are master regulators of intracellular signaling and can directly regulate embryo implantation and endometrial remodeling during murine pregnancy. In immortalized human uterine cells, we have shown that glucocorticoids and estradiol (E₂) coregulate thousands of genes. Recently, glucocorticoids and E₂ were shown to coregulate the expression of Left-right determination factor 1 (LEFTY1), previously implicated in the regulation of decidualization. To elucidate the molecular mechanism by which glucocorticoids and E₂ regulate the expression of LEFTY1, immortalized and primary human endometrial cells were evaluated for gene expression and receptor recruitment to regulatory regions of the LEFTY1 gene. Glucocorticoid administration induced expression of LEFTY1 messenger RNA and protein and recruitment of the glucocorticoid receptor (GR) and activated polymerase 2 to the promoter of LEFTY1. Glucocorticoid-mediated recruitment of GR was dependent on pioneer factors FOXA1 and FOXA2. E₂ was found to antagonize glucocorticoid-mediated induction of LEFTY1 by reducing recruitment of GR, FOXA1, FOXA2, and activated polymerase 2 to the LEFTY1 promoter. Gene expression analysis identified several genes whose glucocorticoid-dependent induction required FOXA1 and FOXA2 in endometrial cells. These results suggest a molecular mechanism by which E₂ antagonizes GR-dependent induction of specific genes by preventing the recruitment of the pioneer factors FOXA1 and FOXA2 in a physiologically relevant model.

Glucocorticoids selectively regulate human endometrial gene expression through cooperation with the pioneer factors FOXA1 and FOXA2, which can be modulated through estrogen receptor activity.

The changes that occur in endometrial structure and function during early pregnancy rely on dynamic spatiotemporal control over the uterine transcriptome (1). Uterine receptivity is dependent on the coordinated expression of many signaling proteins, including chemotactic factors, growth factors, adhesion molecules, and transcription factors. For example, postovulation the human endometrium undergoes a decidualization process driven by progesterone and estrogen, which leads to the induction of prostaglandins, cytokines, and integrins that promote endometrial vascular permeability and attachment of the blastocyst to the uterine wall. The timing of these molecular changes is essential to ensure successful pregnancy, as each discrete stage of pregnancy relies on the success of previous stages. However, the molecular mechanisms governing the stage-specific transcriptional profile in the uterus during pregnancy are not well understood due to overlapping expression patterns or complete infertility in transgenic mouse models (1). Moreover, the mechanisms by which physiological signals are incorporated to regulate reproductive success are not clear.

Transcriptional regulation occurs through many mechanisms, including the targeted recruitment of transcription factors and cofactors (2). The ovarian steroid hormones estrogen and progesterone bind their respective nuclear receptors to coordinate uterine functions by acting as transcription factors (1). Although the importance of the ovarian hormones in uterine physiology is well established, the role of glucocorticoids as reproductive transcriptional regulators is increasingly being recognized (3–5). Glucocorticoid action is mediated by intracellular signaling via the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily of transcription factors (6, 7). Female mice lacking GR in the uterus are subfertile, exhibiting reduced blastocyst implantation and subsequent defects in endometrial decidualization (8). In rodents, exogenous administration of the synthetic glucocorticoid dexamethasone (dex) blocked uterine growth and differentiation and diminished rates of embryo implantation, suggesting that an appropriate balance of glucocorticoid signaling is required for successful pregnancy (9–11).

In vitro studies in immortalized human endometrial cells have shown that glucocorticoids and estradiol (E₂) commonly regulate thousands of genes (12). Regulation of glucocorticoid-induced leucine zipper (GILZ) by glucocorticoids and E₂ was found to involve recruitment of their respective receptors to the same regulatory region of the promoter (13). Presence of estrogen receptor (ER)-α at the glucocorticoid response element (GRE) was correlated with decreased activated polymerase 2 occupancy at the transcriptional start site. Coregulation of gene expression by glucocorticoids and E₂ has also been demonstrated in a variety of other cell types (14–16). Studies in mammary cell lines have shown that glucocorticoids and E₂ work together to reprogram the chromatin landscape and dynamically coregulate the genomic distribution of chromatin pioneer factors (17, 18). Pioneer factors are transcription factors that can penetrate chromatin to facilitate the recruitment of transcription factors and other regulatory proteins (19). GR and ER rely on pioneer factors to facilitate signaling, though it is not understood how pioneer factors contribute to glucocorticoid and estrogen coregulation of gene expression in the uterus (20, 21).

Expression of Left-right determination factor 1 (LEFTY1), a regulator of the NODAL signaling pathway, is regulated by glucocorticoids and estrogen in immortalized human uterine cells (12). LEFTY is temporally regulated in the epithelial and stromal cells of the human endometrium during the menstrual cycle and is believed to be a critical mediator of differentiation (22–24). LEFTY knockdown in human uterine fibroblast cells during decidualization increases the expression of decidual markers and transcription factors essential to decidualization, whereas excess LEFTY expression in mice adversely affects the ability to establish pregnancy and decreases artificial decidualization (25). Levels of LEFTY in the endometrial fluid of infertile women are higher during the receptive phase than fertile women (26). Adverse effects in response to absent or excessive LEFTY levels indicate that expression is precisely regulated for successful pregnancy, and understanding the mechanisms by which this occurs may lead to a better understanding of the signaling networks required for uterine function.

We used immortalized human Ishikawa cells, immortalized human endometrial stromal cells (HESCs), and primary human endometrial stromal cells (ESCs) to evaluate the mechanism of E₂ antagonism of glucocorticoid-induced LEFTY1 induction. Here, we show that pioneer factors FOXA1 and FOXA2 cooperate to facilitate GR recruitment to the LEFTY1 promoter and that E₂ antagonizes glucocorticoid responsiveness by preventing recruitment of GR, FOXA1, and FOXA2. Moreover, gene expression studies indicate that pioneer factors may be critical to glucocorticoid regulation of several genes in immortalized human uterine endometrial cells. The study presented here provides a molecular understanding of the mechanisms governing glucocorticoid action in human endometrial cells.

Materials and Methods

Reagents

RPMI 1640, Dulbecco’s modified Eagle medium (DMEM), and DMEM/Ham F12 were purchased from Invitrogen (Life Technologies, Inc., Carlsbad, CA). Heat inactivated fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO). Charcoal-dextran treated (stripped) heat inactivated FBS was purchased from Gemini Bio-Products (Sacramento, CA). FOXA1, FOXA2, NR3C1, LEFTY1, and nontargeting control (NTC) ON-Target Plus small-interfering RNA (siRNA) were purchased from GE Healthcare Dharmacon (Fisher, Pittsburgh, PA). TaqMan real-time polymerase chain reaction (RT-PCR) primer-probes were purchased from Applied Biosystems (Foster City, CA). Dex (1, 4-pregnadien-9α-fluoro-16α-methyl-11β, 17, 21-triol-3, 20-dione), corticosterone (4-pregnen-11β, 21-diol-3, 20-dione), E₂ (1,3,5[10] estratrien-3,17β-diol), and Mifepristone (RU486; 4, 9-Estradien-17α-Propynyl, 11β-[4-Dimethynylamino]Phenyl-17β-OL-3-One) were purchased from Steraloids (Newport, RI). Dex and corticosterone were prepared in phosphate-buffered saline (PBS). E₂ was prepared in ethanol and dissolved in PBS for an experimental concentration of 10 nM.

Culture of human cells

Cell lines were grown in standard conditions with 5% carbon dioxide. ESCs were prepared from endometrial biopsies as previously described (27) and grown in DMEM/Ham F12 supplemented with 10% fetal calf serum. Twenty-four hours prior to experiments, media were changed to phenol red-free DMEM Ham F12 supplemented with 5% charcoal-stripped fetal calf serum. ESCs were used before the sixth passage for all experiments. HESCs were grown in DMEM supplemented with 10% FBS, 1.0 mM sodium pyruvate, 10 mM HEPES, 0.10 mM MEM nonessential amino acids, and 100 U/mL penicillin/streptomycin (28). Twenty-four hours prior to experiments, cell medium was changed to phenol red-free DMEM containing 10% FBS, 1.0 mM sodium pyruvate, 10 mM HEPES, 0.10 mM MEM nonessential amino acids, and 100 U/mL penicillin/streptomycin. Immortalized human uterine endometrial adenocarcinoma (Ishikawa) cells were maintained in RPMI 1640 supplemented with 5% FBS. Twenty-four hours prior to treatment, cell medium was changed to phenol red-free RPMI 1640 containing 5% stripped FBS. Short tandem repeat analysis was conducted on Ishikawa cells at the DNA Analysis Facility at Yale University to authenticate the cell line.

RNA interference (siRNA)

Cells were plated in six-well plates at ~70% confluency and transfected with 50 nM siRNA against FOXA1, FOXA2, NR3C1 (GR), or LEFTY1 using DharmaFECT transfection reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. The following day, each well was split for experimental end point and to confirm knockdown.

Cell proliferation assay

Cells were plated at a density of 2 × 10⁴ cells per well 1 day after transfection with either NTC or LEFTY1 siRNA. Trypsinized cells were counted 48, 72, and 96 hours after plating with the Countess Automated Cell Counter (Thermo Fisher Scientific) using chamber slides with a 1:1 dilution of cells to Trypan blue stain 0.4% (Thermo Fisher Scientific). The average number of cells per treatment and time point was calculated from 9 to 18 independent experiments.

Quantitative RT-PCR

Total RNA was prepared from cells using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA) with a DNase treatment performed on column. Complementary DNA was synthesized from 100 ng total RNA using the One-Step RT-PCR Universal Master Mix reagent (Applied Biosystems/Thermo Fisher Scientific). Quantitative RT-PCR (qRT-PCR) was performed using the QuantStudio 6 (Applied Biosystems/Thermo Fisher Scientific) or CFX384 thermocycler (BioRad, Hercules, CA) with predesigned primer-probe sets (Applied Biosystems; Supplemental Table 1 ^{(19.8KB, docx)} ). The signal obtained from each gene primer-probe set was normalized to that of the unregulated housekeeping gene peptidylprolyl isomerase B (PPIB). Analysis of nascent LEFTY1 RNA was determined with primer sequences designed to amplify a region spanning an exon-intron boundary, which allowed detection of newly expressed, unprocessed transcripts. All qRT-PCR analysis was determined from at least four biological replicates. Glucocorticoid pathway targets were analyzed using the glucocorticoid signaling human PrimePCR pathway array (BioRad). Heat maps of gene expression were created using the Bio-Rad CFX Manager software.

Western blotting

Ishikawa cells were lysed in Tris glycine sodium dodecyl sulfate (SDS) sample buffer supplemented with 2-mercaptoethanol. Protein concentrations were measured using the Pierce 660 nm protein assay kit (Thermo Fisher Scientific). Equal amounts of protein from cell extracts were separated via SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked with 7.5% skim milk in Tris-buffered saline and incubated overnight with primary antibodies against β-actin, FOXA1, FOXA2, LEFTY1, or GR (Supplemental Table 2 ^{(19.8KB, docx)} ) in 5% milk in Tris-buffered saline-Tween (0.1%). Immunoreactivity was visualized using the Odyssey LI-COR imaging system (LI-COR Biosciences, Lincoln, NE). Protein levels were normalized to β-actin and expressed relative to NTC siRNA samples.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature (RT). After washing with PBS, cells were incubated for 30 minutes with 5% normal goat serum (NGS) at RT. Cells were then incubated overnight at 4°C with antibodies against Ki67 (Supplemental Table 2 ^{(19.8KB, docx)} ) in 3% bovine serum albumin, 0.2% Triton X-100. The next day, cells were washed three times with PBS, then incubated for 20 minutes in 5% NGS. Cells were then incubated in secondary antibody (Supplemental Table 2 ^{(19.8KB, docx)} ) in 5% NGS for 30 minutes at RT. DRAQ5 (Cell Signaling Technologies, Beverly, MA) was added prior to mounting to visualize the nucleus. Images were obtained on a Zeiss LSM780 confocal microscope equipped with a 63X (oil) objective and processed using the Zen 2012 software.

Chromatin immunoprecipitation

Ishikawa cells treated with dex for 0.75 or 1.5 hours were fixed with 1% paraformaldehyde for 10 minutes at RT. Cells were then washed in ice-cold PBS and the cross-linking stopped with 125 nM glycine. Cells were resuspended in cell lysis buffer containing 50 mM HEPES-KOH pH 8.0, 1 mM EDTA, 140 mM NaCl, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors and dounce homogenized. Samples were centrifuged for 10 minutes at 5000 rpm at 4°C and resuspended in shearing buffer containing 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 140 mM NaCl, 1.0% SDS, 0.1% Na Deoxycholate, 1% Triton X-100, and protease inhibitors. Samples were then sonicated using the Fisher Scientific Model 120 Sonic Dismembrator (Thermo Fisher Scientific) at 35% for 6 minutes to obtain 200- to 500-bp fragments. Fragment size was confirmed by separation on an agarose gel. Sheared chromatin was precleared with protein A agarose/salmon sperm DNA (Millipore, Temecula, CA) and immunoprecipitated overnight with antibodies against GR, ER, FOXA1, FOXA2, PolII-S2, or normal rabbit immunoglobulin G (IgG) (Supplemental Table 2 ^{(19.8KB, docx)} ). The DNA complex was then precipitated with protein A magnetic beads (BioRad). After washing and elution, cross-links were reversed and DNA was purified using the QiaQuick PCR Purification kit (Qiagen). The amount of immunoprecipitated DNA was quantified using qRT-PCR with custom-designed primer-probe sets ordered from Integrated DNA Technologies (Coralville, IA; Supplemental Table 3 ^{(19.8KB, docx)} ).

In silico promoter analysis

The JASPAR CORE database, a curated set of profiles derived from published collections of experimentally defined eukaryotic transcription factor binding sites, was used to identify putative binding motifs within the GR binding region of the LEFTY1 gene. Sequences were scanned at a profile score threshold of 90%.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM) from at least four biological replicates. Statistical significance was determined by analysis of variance (ANOVA) with Tukey post hoc analysis. Statistical significance is reported as P < 0.05 (*) or P < 0.01 (**).

Results

Glucocorticoids regulate LEFTY1 expression in primary and immortalized human uterine cell lines

Global gene expression analysis determined that LEFTY1 is a glucocorticoid-responsive gene in immortalized human endometrial cells (12). To establish that glucocorticoid-dependent induction of LEFTY1 represents a physiological cell process occurring in other uterine cell lines and primary cells, Ishikawa, HESCs, and ESCs were treated with vehicle or 100 nM dex for 6 hours. Glucocorticoid treatment significantly induced LEFTY1 expression in all cell types [Fig. 1(a)]. Nascent RNA primers targeting exon-intron boundaries were used to determine whether glucocorticoid treatment altered the transcription of the LEFTY1 gene. After 6 hours of exposure to dex, a significant induction of LEFTY1 nascent RNA was observed in all cell lines [Fig. 1(b)]. Glucocorticoid-mediated induction of LEFTY1 was abolished when Ishikawa, HESC, and primary endometrial cells were pretreated with the GR antagonist RU486 [Fig. 1(c)]. To determine GR transactivation was required for glucocorticoid-mediated induction of LEFTY1, Ishikawa, HESC, and primary endometrial cells were transfected with siRNA against NR3C1, the gene encoding for GR, and treated with and without dex. Knockdown of GR was confirmed by Western blot in both immortalized and primary cells [Fig. 1(d)]. As previously demonstrated (12), glucocorticoid-regulated induction of LEFTY1 messenger RNA (mRNA) was abolished following NR3C1 knockdown in Ishikawa cells. In HESC and primary endometrial stromal cells, glucocorticoids induced LEFTY1 expression, and knockdown of NR3C1 eliminated glucocorticoid induced upregulation of LEFTY1 mRNA [Fig. 1(e)]. Collectively, these data indicate that glucocorticoids act through GR to direct induce the expression of LEFTY1 in multiple uterine cell types.

Figure 1.

Glucocorticoids regulate LEFTY1 expression in immortalized and primary human endometrial cells. (a) LEFTY1 mRNA was measured by qRT-PCR in Ishikawa cells, HESCs, and ESCs following 6 hours of treatment with vehicle (saline) or 100 nM dex. (b) Nascent LEFTY1 mRNA was measured by qRT-PCR in cells treated with vehicle or 100 nM dex. Values were normalized to nascent PPIB and set relative to vehicle. (c) LEFTY1 mRNA was measured by qRT-PCR in Ishikawa cells, HESCs, and ESCs treated first with 1 μM RU486 for 1 hour followed by 6 hours of vehicle or dex. (d) Representative Western blot is provided and GR protein expression in Ishikawa, HESC, and primary endometrial cells transfected with NTC or NR3C1 siRNA has been graphed. Quantified protein expression was normalized to β-actin and expressed relative to GR protein levels in NTC siRNA treatment. (e) LEFTY1 mRNA was measured by qRT-PCR in Ishikawa, HESC, and primary endometrial cells transfected with NTC (white bars) or NR3C1 (black bars) siRNA and treated for 6 hours with vehicle or 100 nM dex. For all qRT-PCR experiments, mRNA levels were normalized to that of PPIB and set relative to vehicle or vehicle-treated NTC siRNA. Bar graphs represent mean ± SEM. *P < 0.05; **P < 0.01 as determined by ANOVA.

E₂ antagonizes glucocorticoid-mediated induction of LEFTY1

To determine the kinetics of glucocorticoid regulation of LEFTY1 expression, Ishikawa cells were treated with the endogenous glucocorticoid cortisol or the synthetic glucocorticoid dex for 6 hours at a range of concentrations representing physiological and pharmaceutical exposure (0 nM to 1000 nM) [Fig. 2(a)]. Cortisol significantly induced LEFTY1 mRNA expression at 1 nM to 1000 nM, with the peak induction for cortisol occurring at 10 nM. Induction of LEFTY1 mRNA by dex was significant at 10 nM to 1000 nM. Expression of LEFTY1 mRNA was maximal following treatment with 100 nM dex.

Figure 2.

Glucocorticoids regulate LEFTY1 expression in Ishikawa cells. (a) Ishikawa cells were treated with corticosterone (dashed line) or dex (black line) at a range of concentrations (0 nM to 1000 nM) for 6 hours. LEFTY1 mRNA expression was evaluated by qRT-PCR. (b) Expression of LEFTY1 mRNA was measured by qRT-PCR in cells treated for 6 hours with vehicle (Veh), 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂. (c) Expression of LEFTY1 mRNA was measured by qRT-PCR in cells treated for 0 to 24 hours with 100 nM dex (black line) or 100 nM dex and 10 nM E₂ (dashed line). (d) Nascent LEFTY1 mRNA was measured by qRT-PCR in cells treated for 0 to 6 hours with 100 nM dex (white bars) or 100 nM dex and 10 nM E₂ (black bars). Values were normalized to nascent PPIB and set relative to 0 hours. (e) Representative Western blot and quantified LEFTY1 protein expression in cells treated for 24 hours with Veh, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂. Quantified protein expression was normalized to β-actin and expressed relative to LEFTY1 protein levels in Veh-treated cells. For all qRT-PCR experiments, mRNA levels were normalized to that of PPIB and set relative to 0 hours or Veh samples. Bar graphs represent mean ± SEM. **P < 0.01 as determined by ANOVA.

Regulation of LEFTY1 mRNA expression is differentially responsive to dex in the presence of E₂ [Fig. 2(b)]. Coregulation of LEFTY1 mRNA by glucocorticoids and E₂ requires GR and ER-α, respectively, but the mechanism by which this occurs has not been fully evaluated (12). To elucidate the kinetics of E₂-mediated antagonism of LEFTY1 induction by glucocorticoids, Ishikawa cells were treated with vehicle, 100 nM dex, or 100 nM dex and 10 nM E₂. E₂ significantly repressed glucocorticoid-regulated induction of LEFTY1 mRNA by 1 hour posttreatment, and antagonism was present at 3, 6, and 24 hours posttreatment [Fig. 2(c)]. Nascent LEFTY1 mRNA increased in response to glucocorticoid treatment, and cotreatment with E₂ antagonized nascent LEFTY1 induction, suggesting that antagonistic regulation of LEFTY1 mRNA occurs at the transcriptional level [Fig. 2(d)]. Western blot analysis showed that LEFTY1 protein levels were upregulated following treatment with dex, and cotreatment of 100 nM dex and 10 nM E₂ blocked glucocorticoid-mediated upregulation [Fig. 2(e)].

GR is recruited to the LEFTY1 promoter and E₂ prevents recruitment

One mechanism by which GR regulates transcription is through DNA association by direct binding at GREs or in complex with other regulatory factors (29). An in silico motif analysis using the JASPAR CORE database discovered two GREs in close proximity within the promoter of the LEFTY1 gene (30). Additionally, transcription factor chromatin immunoprecipitation (ChIP)–sequencing data from ENCODE with Factorbook motifs reposited in the University of California, Santa Cruz (UCSC) genome browser identified a putative GR binding region 1967 to 2322 bp upstream of the LEFTY1 transcriptional start site. ChIP assays were performed on Ishikawa cells treated with vehicle or glucocorticoids to determine whether GR is recruited to the in silico identified GREs or the GR binding region identified in the UCSC genome browser. Dex treatment induced minimal recruitment of GR to the region encompassing the GREs [Fig. 3(a)] but robustly induced recruitment of GR to the GR binding region identified in the UCSC genome browser [Fig. 3(b)]. Strong recruitment of GR to the GR binding region was also observed in the primary endometrial cells [Fig. 3(c)]. As a positive control, glucocorticoid-induced recruitment of GR to a previously identified nuclear factor κB site in the interleukin 8 gene (31) was evaluated 1.5 hours after treatment and found to be significantly enriched following dex treatment in Ishikawa cells [Fig. 3(d)] and primary endometrial cells [Fig. 3(e)].

Figure 3.

Glucocorticoid-induced GR binding to the LEFTY1 promoter is blocked in the presence of E₂. (a and b) A schematic representation of a GRE identified in the LEFTY1 promoter using the (a) JASPAR database and a putative GR binding region located 1967 bp upstream of the transcriptional start site of LEFTY1 identified in the (b) UCSC genome browser. Ishikawa cells were treated with 100 nM dex for 0, 0.75, or 1.5 hours, and ChIP assays were performed with antibodies against IgG (white bars) or GR (black bars). Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the (a) GRE and (b) GR binding region. (c) ChIP assays were performed in human primary endometrial cells, and coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the GR binding region. Coimmunoprecipitated DNA was analyzed for a nuclear factor κB site in the interleukin 8 promoter in (d) Ishikawa cells and (e) primary endometrial cells as a positive control for GR ChIP. (f and h) ChIP assays were performed with antibodies to IgG and (d) GR, (e) PolII, or (f) ER-α in Ishikawa cells treated for 1.5 hours with vehicle (Veh), 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to (f and h) the putative GR binding region and (g) the transcriptional start site (TSS). (i) ChIP assays were performed with antibodies against IgG (white bars) or ER-α (black bars) in Ishikawa cells treated for 1.5 hours with Veh 10 nM E₂. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to an ERE in Trefoil factor 1 (Tff1). All ChIP data were plotted relative to input DNA.

To investigate the effect of E₂ treatment on glucocorticoid-induced binding of GR to the LEFTY1 promoter, ChIP assays were conducted on Ishikawa cells treated with vehicle, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂. Glucocorticoid-induced recruitment of GR to the LEFTY1 promoter was absent in the presence of E₂ [Fig. 3(f)]. Glucocorticoid-induced recruitment of activated RNA polymerase II (PolII) to the LEFTY1 transcriptional start site was also absent following cotreatment with E₂ [Fig. 3(g)]. However, treatment with glucocorticoids, E₂, or both glucocorticoids and E₂ did not lead to ER-α recruitment to the GR binding region of the LEFTY1 promoter [Fig. 3(h)]. As a positive control, E₂-induced recruitment of ER-α to a validated estrogen response element (ERE) in Trefoil factor 1 (Tff1) was evaluated 1.5 hours after treatment, and ER recruitment was found to be significantly enhanced at this ERE following E₂ treatment [Fig. 3(i)] (13). This finding suggests that E₂ does not antagonize LEFTY1 induction through competitive binding of ER-α at the GR binding region. Alternatively, ER-α may regulate other proteins that mediate GR binding. To identify other transcription factors that could potentially bind the GR binding region, an in silico motif analysis of the sequence was performed (Table 1). Twelve putative motifs were identified using a profile score threshold of 90% and a score threshold of 9.5. The motif for the pioneer factor FOXA1 was identified in the GR binding region.

Table 1.

In Silico Motif Analysis for the GR Binding Region of LEFTY1

Motif	Score	Predicted Site Sequence
SOX9	12.651	CCATTGTTT
E2F6	11.871	GGGTGGGAAAC
VSX2	11.037	TTAATTAT
VSX1	10.969	TTAATTAT
VAX1	10.599	TTAATTAT
VAX2	10.321	TTAATTAT
ALX3	10.032	TTTAATTATG
ISX	9.948	TTAATTAT
LMX1A	9.829	TTAATTAT
FOXA1	9.770	GTGATGTTTGTTCAG
FOXO3	9.691	GGAAACAA
PRRX1	9.638	TTAATTAT

FOXA1 is recruited to the GR binding region in the LEFTY1 promoter

A comparison of the putative binding factors with those annotated in the transcription factor ChIP-sequencing data identified FOXA1 as a candidate cofactor, with binding of FOXA1 shown 2022 to 2330 bp upstream of the LEFTY1 transcriptional start site in the UCSC genome browser. ChIP assays were performed following vehicle or glucocorticoid treatment to determine whether FOXA1 is associated with GR binding to the LEFTY1 promoter [Fig. 4(a)]. FOXA1 was strongly enriched at the GR binding region of the LEFTY1 promoter when cells were exposed to glucocorticoids for 1.5 hours. ChIP assays were then conducted on cells treated with vehicle, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂ to determine whether E₂ treatment altered glucocorticoid-induced FOXA1 recruitment [Fig. 4(b)]. Similar to the effect on GR recruitment, E₂ blocked glucocorticoid-induced recruitment of FOXA1 to the LEFTY1 promoter.

Figure 4.

Glucocorticoids induce recruitment of FOXA1 to the GR binding region in the LEFTY1 promoter. (a) ChIP assays were performed using antibodies against IgG (white bars) or FOXA1 (black bars) in cells treated with 100 nM dex for 0, 0.75, or 1.5 hours. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the putative GR binding region of LEFTY1. (b) ChIP assays were performed using antibodies against IgG (white bars) or FOXA1 (black bars) in cells treated with vehicle (Veh), 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂ and analyzed by qRT-PCR using primers to the GR binding region of LEFTY1. (c) Expression of FOXA1 mRNA was measured by qRT-PCR in cells transfected with NTC or FOXA1 siRNA. Representative Western blot and quantified FOXA1 protein expression in cells transfected with NTC or FOXA1 siRNA. Quantified protein expression was normalized to β-actin and expressed relative to FOXA1 protein levels in NTC siRNA treatment. (d) Expression of LEFTY1 mRNA was measured by qRT-PCR in cells transfected with NTC (white bar) or FOXA1 (black bar) siRNA and treated for 6 hours with either Veh or dex. Cells transfected with either NTC (white bar) or FOXA1 (black bar) siRNA were treated with Veh or 100 nM dex for 1.5 hours and assayed by ChIP for recruitment of (e) GR or (f) FOXA1 to the GR binding region in the LEFTY1 promoter. For all qRT-PCR experiments, mRNA levels were normalized to that of PPIB and set relative to Veh or Veh-treated NTC siRNA. All ChIP data were plotted relative to input DNA. Bar graphs represent mean ± SEM. *P < 0.05; **P < 0.01 as determined by ANOVA.

To investigate whether FOXA1 recruitment is important for glucocorticoid regulation of LEFTY1, FOXA1 mRNA expression was knocked down in Ishikawa cells, and knockdown was confirmed by qRT-PCR and Western blot analysis [Fig. 4(c)]. LEFTY1 gene expression was measured in cells transfected with NTC or FOXA1 siRNA and treated with vehicle, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂. Knockdown of FOXA1 reduced glucocorticoid-mediated induction of LEFTY1 mRNA by approximately one-third [Fig. 4(d)]. Although this reduction was significant, mRNA expression of LEFTY1 in glucocorticoid-treated FOXA1 knockdown cells remained six times higher than that of vehicle-treated cells. Additionally, FOXA1 knockdown did not alter glucocorticoid-induced GR binding to the LEFTY1 promoter [Fig. 4(e)]. To evaluate the specificity of the FOXA1 antibody, ChIP experiments were repeated in Ishikawa cells transfected with FOXA1 siRNA [Fig. 4(f)]. Following FOXA1 knockdown, FOXA1 was no longer found recruited to the LEFTY1 promoter with dex treatment. These data suggest that FOXA1 contributes to glucocorticoid regulation of LEFTY1 expression, although other factors may also be involved.

FOXA1 and FOXA2 are necessary for glucocorticoid-mediated induction of LEFTY1

FOXA2 is a member of the forkhead box protein family that shares structural similarity to and several redundant roles with FOXA1 (32–35). Transcription factor ChIP-sequencing data in the ENCODE database identified a FOXA2 binding site 2093 to 2240 bp upstream of the LEFTY1 transcriptional start site, overlapping with the FOXA1 and GR binding region. To evaluate whether FOXA2 performs a redundant role with FOXA1 at the GR binding region of the LEFTY1 promoter, ChIP assays were conducted on cells transfected with NTC or FOXA1 siRNA and treated with vehicle or 100 nM dex [Fig. 5(a)]. In the presence of FOXA1, dex did not induce recruitment of FOXA2 to the GR binding region. However, following knockdown of FOXA1, dex induced recruitment of FOXA2 to the GR binding region. This finding suggests that FOXA2 may cooperate with FOXA1 to promote glucocorticoid-dependent GR binding to the LEFTY1 promoter. The recruitment of FOXA2 to the GR binding region in LEFTY1 was evaluated in cells transfected with FOXA1 siRNA and treated with vehicle, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂ [Fig. 5(b)]. Cotreatment of glucocorticoids with E₂ blocked glucocorticoid-induced recruitment of FOXA2 to the LEFTY1 promoter. To determine the role of FOXA2 in glucocorticoid-mediated regulation of LEFTY1, FOXA2 mRNA expression was knocked down in Ishikawa cells, confirmed by qRT-PCR and Western blot analysis [Fig. 5(c)]. Expression of LEFTY1 was measured in cells transfected with NTC or FOXA2 siRNA and treated with vehicle, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂ [Fig. 5(d)]. Silencing of FOXA2 did not completely abrogate glucocorticoid-induced LEFTY1 mRNA expression but did decrease it by approximately one-third, similar to glucocorticoid-mediated levels of LEFTY1 found following FOXA1 knockdown.

Figure 5.

Glucocorticoids induce FOXA2 recruitment to the GR binding region in the absence of FOXA1. (a) ChIP assays were performed using antibodies against IgG (white bars) or FOXA2 (black bars) in cells transfected with NTC or FOXA1 siRNA and treated for 1.5 hours with vehicle (Veh) or 100 nM dex. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the putative GR binding region of LEFTY1. (b) ChIP assays were performed using antibodies against IgG (white bars) or FOXA2 (black bars) in cells transfected with FOXA1 siRNA and treated for 1.5 hours with Veh, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the putative GR binding region of LEFTY1. (c) Expression of FOXA2 mRNA was measured by qRT-PCR in cells transfected with NTC or FOXA2 siRNA. Representative Western blot image is provided and quantified FOXA1 protein expression is reported for cells transfected with NTC or FOXA2 siRNA. Quantified protein expression was normalized to β-actin and expressed relative to FOXA2 protein levels in NTC siRNA treatment. (d) Expression of LEFTY1 mRNA was measured by qRT-PCR in cells transfected with either NTC (white bars) or FOXA2 (black bars) siRNA and treated for 6 hours with Veh, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂. For all qRT-PCR experiments, mRNA levels were normalized to that of PPIB and set relative to Veh-treated NTC siRNA. ChIP data were plotted relative to input DNA. Bar graphs represent mean ± SEM. *P < 0.05; **P < 0.01 as determined by ANOVA.

To evaluate whether glucocorticoid-dependent induction of LEFTY1 requires both FOXA1 and FOXA2, expression of LEFTY1 was measured in cells transfected with NTC or FOXA1 and FOXA2 (FOXA1/2) siRNA and treated with vehicle, 100 nM dex, 10 nM E₂, or 100 nM dex and 10 nM E₂ [Fig. 6(a)]. Glucocorticoid-mediated induction of LEFTY1 through GR requires FOXA1 and FOXA2, as knockdown of both FOXA1 and FOXA2 abolished LEFTY1 induction by dex. Induction of LEFTY1 in primary endometrial cells by glucocorticoids also requires FOXA1 and FOXA2 [Fig. 6(b)]. Additionally, glucocorticoid-induced GR recruitment to the LEFTY1 promoter was absent in the FOXA1/2 knockdown cells compared with cells transfected with NTC siRNA, which demonstrated robust recruitment [Fig. 6(c)]. Glucocorticoid-induced activated PolII recruitment to the LEFTY1 transcriptional start site was also absent in FOXA1/2 knockdown cells compared with the NTC control [Fig. 6(d)]. These findings indicate that FOXA1 and FOXA2 regulate GR recruitment to the LEFTY1 promoter and subsequent upregulation of LEFTY1.

Figure 6.

Glucocorticoid signaling requires FOXA1 and FOXA2. (a and b) Expression of LEFTY1 mRNA was measured by qRT-CPR in (a) Ishikawa and (b) primary endometrial cells transfected with NTC (black bars) or FOXA1 and FOXA2 (FOXA1/2; black bars) siRNA. mRNA levels were normalized to that of PPIB and set relative to vehicle (Veh)-treated NTC siRNA samples. (c) ChIP assays were performed with antibodies against IgG (white bars) or GR (black bars) in cells transfected with either NTC or FOXA1/2 siRNA. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the putative GR binding region of LEFTY1. (d) ChIP assays were performed with antibodies against IgG (white bars) or PolII-S2 (black bars) in cells transfected with either NTC or FOXA1/2 siRNA. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the putative GR binding region of LEFTY1. (e and f) Total RNA from cells transfected with NTC or FOXA1/2 siRNA and treated for 6 hours with Veh or 100 nM dex were evaluated by qRT-PCR using the Glucocorticoid Signaling Pathway Array from BioRad. mRNA levels were normalized to that of TATA binding protein (TPB). (e) Heat map depicts genes in which glucocorticoid-induced expression was significantly attenuated by FOXA1/2 knockdown. Relative mRNA expression was graphed for TNF-α. (f) Heat map depicts eight genes in which glucocorticoid-induced expression is not altered by FOXA1/2 knockdown. Relative mRNA expression was graphed for Period circadian clock 1 (PER1). All ChIP data were plotted relative to input DNA. Bar graphs represent mean ± SEM. *P < 0.05; **P < 0.01 as determined by ANOVA.

To investigate whether FOXA1 and FOXA2 are generally required for glucocorticoid signaling in immortalized human endometrial cells, NTC or FOXA1/2 siRNA transfected cells were treated for 6 hours with vehicle or 100 nM dex, and mRNA were evaluated using a Glucocorticoid Signaling Pathway Array plate (Supplemental Fig. 1 ^{(101.6KB, pptx)} ). Glucocorticoid-mediated upregulation was lost following FOXA1/2 knockdown for eight genes: SLC10A6, platelet-derived growth factor receptor β (PDGFRB), SplA/ryanodine receptor domain and SOCS box containing 1 (SPSB1), IL-10, tumor necrosis factor-α (TNF-α), Aquaporin 1 (AQP1), signal transducer and activator of transcription 5A (STAT5A), and DNA damage inducible transcript 4 (DDIT4) [Fig. 6(e)]. However, several classic glucocorticoid target genes were unaffected by FOXA1/2 knockdown, including PER1, BCL6, FKBP5, NFKB1, KLF9, TSC22D3, USP2, and FOSL2 [Fig. 6(f)]. Of the glucocorticoid-responsive genes that require the presence of FOXA1 and/or FOXA2 for their induction, several contained adjacent or overlapping GR and FOXA1 or FOXA2 binding sites (as determined by the UCSC genome browser), potentially identifying a regulatory region where the pioneer factors FOXA1 and FOXA2 are associated with GR binding [Fig. 7(a)]. Recruitment of GR to these putative GR binding regions was evaluated. Dex treatment induced recruitment of GR to the GR binding region identified in the UCSC genome browser of SPSB1, STAT5A, and DDIT4 of cells transfected with NTC siRNA, and knockdown of both FOXA1 and FOXA2 abolished GR recruitment to these genes [Fig. 7(b)].

Figure 7.

FOXA1 and FOXA2 mediate GR binding at select genomic regions. (a) Schematic representation of GR, FOXA1, and FOXA2 binding sites either overlapping or in close proximity to the chromosomal location of genes whose glucocorticoid-mediated expression is dependent on FOXA1/2 (see Fig. 7). Genes represented include SPSB1, STAT5A, TNF-α, and DDIT4, and PDGFRB. Binding sites were identified using transcription factor ChIP-sequencing from Encode with Factorbook motifs in the UCSC genome browser. (b) ChIP assays were performed with antibodies against IgG (white bars) or GR (black bars) in cells transfected with either NTC or FOXA1/2 siRNA. Coimmunoprecipitated DNA was analyzed by qRT-PCR using primers to the putative GR binding region of SPSB1 (downstream of the TTS), DDIT4 (immediately upstream of the TSS), and STAT5A (intragenic) and set relative to input DNA. Bar graphs represent mean ± SEM. **P < 0.01 as determined by ANOVA.

LEFTY1 modulates proliferation in immortalized human endometrial cells

LEFTY1 exhibits temporal expression in the human endometrium, with maximal expression occurring during the late secretory and menstrual phases of the cycle when endometrial proliferation has ceased (22). To assess the physiological relevance of LEFTY1 mRNA regulation in the endometrium, Ishikawa cells were transfected with siRNA against LEFTY1 or a NTC pool and proliferation was quantified. LEFTY1 knockdown was verified by qRT-PCR and Western blot analysis 48 hours following transfection [Fig. 8(a)]. Knockdown of LEFTY1 significantly increased cell proliferation 72 hours following plating compared with the NTC pool [Fig. 8(b)]. Cells transfected with LEFTY1 siRNA also expressed higher levels of the cell proliferation marker Ki67 [Fig. 8(c)]. Treatment with dex reduced cell proliferation rates, which was alleviated following LEFTY1 knockdown [Fig. 8(d)]. These data suggest that glucocorticoids regulate proliferation in immortalized human endometrial cells through a LEFTY1-dependent mechanism.

Figure 8.

LEFTY1 regulates proliferation in immortalized human endometrial cells. (a) Ishikawa cells were transfected with NTC or LEFTY1 siRNA. mRNA was harvested 72 hours following transfection, and levels of LEFTY1 mRNA were measured by qRT-PCR. Values were normalized to PPIB and set relative to NTC siRNA samples. Bar graphs represent mean ± SEM. (b) Cells transfected with NTC (solid line) or LEFTY1 (dash line) siRNA were plated at a concentration of 2 × 10⁴ cells per well. Cells were counted 48, 72, and 96 hours postplating with the Countess Automated Cell Counter. Time points represent the average ± SEM for 11 to 19 independent experiments. (c) Representative immunofluorescence staining of Ishikawa cells with Ki67 antibodies 48 hours following transfection with NTC or LEFTY1 siRNA. Nuclear visualization is indicated by DRAQ5. (d) Cells treated with vehicle (solid line), 100 nM dex (dash line), or transfected with LEFTY1 siRNA and treated with 100 nM dex (dotted line) were plated at a concentration of 2 × 10⁴ cells per well. Cells were counted 48, 72, and 96 hours postplating with the Countess Automated Cell Counter. Time points represent the average ± SEM for 5 to 13 independent experiments. *P < 0.05; **P < 0.01 as determined by ANOVA.

Discussion

Stress-induced glucocorticoid excess leads to infertility, but very little is known about the mechanisms by which glucocorticoid signaling in the uterus contributes to fertility or infertility (4). Microarray analysis has determined that glucocorticoids and E₂ commonly regulate a wide range of genes in immortalized human endometrial cells, including some which are antagonistically regulated (12). The promoters of these genes are enriched for GR and ER binding sites, suggesting that antagonistically regulated genes may be direct targets of GR and ER (12). The current study examined the mechanism by which glucocorticoids and E₂ antagonistically regulate the expression LEFTY1. Gene expression analysis verified that glucocorticoid-regulated induction of LEFTY1 is abrogated in the presence of E₂. Rather than promoting competitive binding of ER to the GR binding site in the LEFTY1 promoter, E₂ antagonizes glucocorticoid signaling by inhibiting recruitment of GR, FOXA1, and FOXA2 to the promoter and preventing activated PolII recruitment to the transcriptional start site. FOXA1 and FOXA2 are considered pioneer factors, meaning that they penetrate closed chromatin to increase the accessibility of transcription factors to DNA binding sites (19, 36). FOXA1 and FOXA2 promote GR binding to the LEFTY1 promoter in a cooperative fashion, and knockdown of both is sufficient to abolish glucocorticoid-induced recruitment of GR to the LEFTY1 promoter. Recent studies have identified a role for FOXA2 in the murine uterus during early pregnancy. Conditional ablation of FOXA2 in the mouse uterus causes disrupted blastocyst implantation and an inability to respond to artificial induction of the decidual response with a severe reduction in the number of endometrial glands (37). The phenotype of the uterine-specific FOXA2 deficient mice partially overlaps with that of the uterine-specific GR deficient mouse, potentially identifying cooperatively regulated genes, which are essential for early stages of pregnancy (8). No studies have yet examined the direct role of FOXA1 in the uterus. However, FOXA1’s role in promoting chromatin binding of steroid receptors is well-established in mammary and prostate cells and may also be relevant in the uterus (36).

Although our results show that FOXA1 and FOXA2 are required for glucocorticoid-dependent binding of GR to the LEFTY1 promoter, it is still unclear how activation of ER-α by E₂ abrogates FOXA1 and FOXA2 enrichment at the promoter. A recent study suggests that FOXA1 and nuclear receptors such as ER-α and GR can reciprocally promote DNA binding for each other through a mechanism called dynamic assisted loading, whereby one transcription factor transiently binds DNA and alters the chromatin landscape to promote recruitment of the other at previously inaccessible sites (18). Estrogen-dependent activation of ER-α may deplete the pool of available FOXA1 and FOXA2, thereby inhibiting the ability of FOXA1 and FOXA2 to modulate GR binding. E₂ may also promote the induction or activity of an unidentified cofactor that prevents FOXA1 and FOXA2 binding to chromatin.

In addition to LEFTY1, gene expression analysis identified eight other classic glucocorticoid responsive genes for which knockdown of FOXA1 and FOXA2 inhibited glucocorticoid-regulated induction. Analysis of reposited ChIP-sequencing data identified GR binding sites overlapping with FOXA1 or FOXA2 binding sites for five of these genes, including TNF-α, SPSB1, STAT5A, DDIT4, and PDGFRB. Dependence on FOXA1 and FOXA2 has not previously been reported for these classic glucocorticoid-responsive genes. Of the eight glucocorticoid-regulated genes, TNF-α, AQP1, and DDIT4 are antagonistically regulated by E₂.

Numerous studies have demonstrated a significant role for the proinflammatory cytokine TNF-α in the uterus. TNF-α upregulation in the uterus following endometrial biopsy has been shown to recruit immune cells that are correlated with improved endometrial receptivity (38). However, aberrant TNF-α activation specifically inhibits trophoblast migration and invasion in vitro and may have pleiotropic effects on the placenta and endometrium (39–43). Aberrant TNF-α expression is also associated with pregnancy complications, such as recurrent spontaneous abortions, preeclampsia, preterm labor, and endometriosis, suggesting that precise regulation of TNF-α expression is required for successful pregnancy (39). SPSB1 and STAT5A are also involved in regulating the immune response (44, 45). SPSB1 upregulates inducible nitrous oxide synthase in response to pathogen recognition by toll-like receptor proteins (44), and STAT5A promotes development of T-cells, B-cells, and natural killer cells (46, 47). Moreover, STAT5 has been implicated in prolactin signaling in the pregnant decidua (48). Although the role of these proteins in the uterus is not well understood, the immune response is integral to successful pregnancy, contributing to adhesion and implantation of the blastocyst, development of immune tolerance to fetal antigens, and protection of the fetus from pathogenic agents (49, 50). Moreover, glucocorticoid signaling was recently demonstrated to be essential for the uterine immune response during early pregnancy (8). Dysregulation of immune response genes by increased levels of glucocorticoids may contribute to stress-induced infertility.

AQP1, a membrane protein that functions as a water channel, has been identified in the nonpregnant endometrium where it is thought to play a role in the water imbibition response to hormones, and mice deficient in AQP1 channels accumulate excessive amniotic fluid during pregnancy (51). DDIT4, also known as Regulated in Development and DNA Damage Response 1 (REDD1), plays an important role in the cellular response to hypoxia and energy stress by negatively modulating signaling of mTOR, a serine-threonine kinase that is essential for cellular growth and development (52–54). In women, administration of the antiprogestin/antiglucocorticoid mifepristone following ovulation causes the endometrium to become unreceptive and is associated with significant changes in gene expression, including repression of DDIT4 mRNA (55). Interestingly, expression of mTOR is present in the endometrial stroma and vital to successful implantation (56). Regulation of mTOR signaling by mediating DDIT4 expression may represent another mechanism by which glucocorticoids regulate fertility.

PDGFRB is an established marker of endometrial mesenchymal stem cells (57). In a recent study, hierarchical clustering analysis of human endometrial cells found that the transcriptome of PDGFRB-positive endometrial mesenchymal stem cells was similar to endometrial fibroblasts and distinct from stromal fibroblasts, suggesting that PDGFRB-positive human endometrial mesenchymal stem cells may be precursors to endometrial stromal fibroblasts (58). Endometrial mesenchymal stem cells expressed pericyte markers, as well as genes involved in steroid hormone responses, angio- and vasculogenesis, cell communication, and immunomodulation, suggesting a role in endometrial vascular remodeling. Although the specific role of PDGFRB in pregnancy has not yet been determined, data from several animal models suggest that PDGFRB contributes to regulation of embryonic development, cell proliferation, survival, differentiation, chemotaxis, and migration (59).

The Centers for Disease Control and Prevention reports that overall fertility and reproductive rates have declined by ~10% in US women over the last 25 years (60). The establishment and maintenance of pregnancy rely upon complex signaling pathways that are temporally and spatially restricted. Given its integral role in pregnancy and in the regulation of several pregnancy-related genes, the glucocorticoid signaling pathway is likely required to support uterine functions during pregnancy. This study demonstrates a molecular mechanism by which E₂ antagonizes glucocorticoid-mediated induction of LEFTY1 in human immortalized endometrial cells by preventing recruitment of GR and the pioneer factors FOXA1 and FOXA2 to the LEFTY1 promoter. Moreover, FOXA1 and FOXA2 are required for glucocorticoid regulation of several genes in immortalized human endometrial cells, which has not been previously reported.

Appendix.

Antibody Table

Peptide/Protein Target	Antigen Sequence (if Known)	Name of Antibody	Manufacturer, Catalog No.	Species Raised In; Monoclonal or Polyclonal	Dilution Used	RRID
FOXA2			Abcam, ab108396	Rabbit; polyclonal	1:1000 Western	AB_10863255
FOXA1			Abcam, ab23738	Rabbit; polyclonal	1:1000 Western	AB_2104842
RNA-polymerase 2 (phospho S2)			Abcam, ab5095	Rabbit; polyclonal	1:175 ChIP	AB_304749
Ki67			Cell Signaling Technologies, 12202	Rabbit; monclonal	1:400 IF	AB_2620142
GR			Cell Signaling Technologies, 3660S	Rabbit; monclonal	1:1000 Western; 1:175 ChIP	AB_11179215
IgG			EMD Millipore, 12-370	Rabbit; polyclonal	1:700 ChIP	AB_145841
Actin			EMD Millipore, MAB1501	Mouse; monoclonal	1:10,000 Western	AB_2223041
ER-α			Santa Cruz Biotechnology, sc-543	Rabbit; polyclonal	1:175 ChIP	AB_631471
FOXA1			Santa Cruz Biotechnology, sc-6553	Goat; polyclonal	1:175 ChIP	AB_2104865
FOXA2			Santa Cruz Biotechnology, sc-6554	Goat; polyclonal	1:175 ChIP	AB_2262810

Abbreviation: RRID, Research Resource Identifier.