A Gata2-Dependent Transcription Network Regulates Uterine Progesterone Responsiveness and Endometrial Function.

Central to women's health is the regulation of uterine function by the ovarian steroid hormones, estradiol (E2) and progesterone (P4), acting through their receptors, the estrogen receptor (ESR1) and progesterone receptor (PGR). PGR is critical for uterine function as shown in the uteri of Pgr -null (PRKO) female mice that are unable to support embryo implantation, undergo decidualization, or inhibit E2 induced endometrial epithelial proliferation with P4 ( Lydon et al., 1995 ). Identifying modifier genes of PGR action and elucidating their interaction will facilitate a comprehensive understanding of mechanisms of P4 actions. We have identified the transcription factor GATA Binding Protein 2 ( Gata2 ) as a potential modifier of uterine P4 signaling ( Rubel et al., 2012a ). Genetic ablation of Gata2 regulates many biological processes, such as hematopoietic cell development ( Tsai et al., 1994 ), vascular integrity ( Johnson et al., 2012 ), adipocyte differentiation ( Tong et al., 2000 ) and pituitary function ( Charles et al., 2006 ). Gata2 is expressed in the epithelium and stroma of the uterus and the epithelial expression coincides with that of Pgr ( Rubel et al., 2012a ). Gata2 has been previously described as coordinating the transcriptional activity of other nuclear receptors including PGR ( Bohm et al., 2009 ; Magklara and Smith, 2009 ; Nagayama et al., 2008 ). These findings suggest GATA2 maybe a modifier of P4 signaling in the uterus through interaction with PGR on the chromatin of P4 target genes. We addressed the role of Gata2 by using the Pgr Cre mouse model to ablate Gata2 in cells expressing Pgr in all compartments of the uterus ( Soyal et al., 2005 ). Mice with Gata2 ablation in the uterus were infertile, lacking both the ability to allow embryo implantation and subsequent uterine stromal decidualization. Importantly, the expression of P4 target genes and Pgr itself were deregulated. Combining cistromic analysis with transcriptomic analysis, we showed that most P4 regulated genes contained both PGR and GATA2 chromatin occupancy demonstrating a cooperative relationship between the two factors in controlling P4 mediated transcription in the mouse uterus. Gata2 ablated uteri showed alterations in epithelial morphology resulting in a transition from a simple epithelium to a stratified squamous epithelium with increased TRP63 expression. Lastly, we present evidence that supports a conservation of the GATA2 - PGR regulatory network in human uteri.

Mice were maintained in the designated animal care facility at Baylor College of Medicine (BCM) according to the Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of laboratory animals. All animal experiments were performed in accordance with an IACUC approved protocol. Detailed description of fertility assay, superovulation, embryo implantation assessment and artificial decidualization are listed in supplemental information . Detailed sample preparation can be found in the supplemental information . All experiments were performed in triplicate with independent pools of RNA. The Partek Genomics Suite 6.6 software (Partek Inc., St. Louis, MO) was utilized to process raw data from CEL files. The Robust Multichip Analysis (RMA) algorithm with quantile for normalization and log2 transformation was applied to generate signal values of all samples. The one-way ANOVA model was used to compare expression profiles from different groups. Differentially expressed genes were defined using the filters of ANOVA unadjusted p value 1.3. GATA2 and Input ChIPs were performed by Active Motif, Inc. (Carlsbad, CA) on mouse uteri treated with P4 and with GATA2 antibody (sc-9008, Santa Cruz) as described in supplemental information . The model-based analysis of ChIP-sequencing (MACS) peak finding algorithm was used to normalize ChIP against Input control ( Zhang et al., 2008 ). Specifically, a P value of 10 −10 was used with this software to identify ChIP peaks in this work by comparing them to input. Genes associated with intervals were assessed using two increasingly less stringent requirements; if it was within 10 kb and 25 kb upstream or downstream of a gene, it was counted. Analysis of enriched motifs and CEAS were performed using the Cistrome Analysis Pipeline software ( http://cistrome.org/ap/ ) under default settings ( Liu et al., 2011 ) or using the HOMER ( Heinz et al., 2010 ). For gene functional classifications, the web application tool Database for Annotation, Visualization, and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov/ ) running default settings was used ( Huang da et al., 2009 ). The publicly available human recurrent implantation failure (RIF) array dataset GSE58144 was scored for manifestation of the mouse model-derived GATA2 signature, using published methods ( Qin et al., 2013 ). In brief, a gene signature score (t-score) was defined for each human sample as the 2-sided t-statistic comparison of the high GATA2-signature with the low GATA2-signature genes’ expression profile. Where multiple probes in GSE58144 referred to the same gene, the probe with the highest variation was chosen to represent the gene. For each gene, the profiles were centered to the median across samples. Expression pattern was displayed as heatmap using Partek Genomics Suite 6.6 software. To test the relationships among variables proposed in the research models, structural equation modeling was conducted to indicate the strength of influence among variables by getting an overall fit of model with the data. Goodness-of-fit tests were performed to determine whether the research models should be accepted or rejected. Models were analyzed using Mplus software version 7.11 (Muthen & Muthen, Los Angeles, CA). Based on the research models, the significance of direct and indirect relationships among variables was examined. All observed data were included when fitting the models. There was no missing data in the dataset. The fit of the models was assessed using several methods including the root mean square error of approximation (RMSEA), along with a 90% confidence interval, the standard root mean square residual (SRMR), the Comparative Fit Index (CFI), and the Tucker-Lewis Fit Index (TLI) to determine the extent to which the relationships existing in the data are consistent with those proposed by the models ( Hu and Bentler, 1998 , 1999 ; MacCallum et al., 1996 ). To interpret the RMSEA, the general rule of thumb is that values .10 indicate poor fit ( MacCallum et al., 1996 ). For both CFI and TLI, a value of 1 indicates perfect fit and the values >.9 indicate adequate fit ( Hu and Bentler, 1998 , 1999 ). Also, values <.08 for SRMR indicate a very good fit between the model and the data. Alkaline Phosphatase Activity Assay was performed on frozen sections. Immunohistochemistry analysis was carried out in paraformaldehyde fixed, paraffin sections with PGR (A0098, Dako) and TP63 (sc-8431, Santa Cruz) antibodies. Detail information is in the supplemental information . Quantitative real-time PCR was performed with Taqman probes listed in supplemental Table 1 . Statistical analyses were performed using t test or one-way ANOVA followed by Tukey's post hoc multiple range test with the Instat package from GraphPad (GraphPad Software, La Jolla, CA). Western blot analysis was carried out with PGR antibody (sc-7208, Santa Cruz). Human endometrial epithelial cells were utilized for transient transfection assays. Detailed descriptions are listed in supplemental information .

We first examined the correlation between GATA2 and PGR in the human endometrium. GATA2 and PGR mRNA levels are positively correlated in human endometrial tissues from 3 independent cohorts, GSE4888 , GSE58144 and GSE51981 ( Fig. 1 A and B ), supporting a conservation of the expression between mouse and human. Given this inference and the role of GATA2 in transcription regulation, we hypothesized that GATA2 may regulate P4 signaling through modulating PGR in uterus. To test this hypothesis, we crossed mice with a floxed Gata2 allele ( Gata2 f/f ) with the Pgr cre mouse to ablate Gata2 in PGR positive cell lineages ( Pgr cre ; Gata2 f/f , Gata2 d/d ) ( Charles et al., 2006 ; Soyal et al., 2005 ). Gata2 deletion was confirmed by Quantitative RT-PCR (qRT-PCR) ( Fig. 1C ). Expression of other Gata genes did not show a compensatory increase in expression in Gata2 d/d uteri ( Fig. 1C ). In fact, Gata2 is the most abundant Gata factor in uterus with its mRNA levels at least 200-fold more than the second-highest expressing Gata factor, Gata6 , ( Fig. 1C ). The fertility of Gata2 d/d mice was determined after a six-month long breeding trial. Six Gata2 f/f females delivered total 251 pups in 33 liters while all 6 Gata2 d/d mice failed to generate any offspring, demonstrating that the Gata2 d/d mice were infertile. To determine the cause of the infertility, Gata2 d/d and Gata2 f/f mice were analyzed on day 5.5 of pregnancy to assess if normal embryo implantation occurred. The Gata2 d/d mice showed no embryo implantation sites compared to that of Gata2 f/f that averaged 7 to 8 sites per mouse ( Fig. 1D, 1E and Table 1 ), demonstrating that loss of Gata2 function leads to failure of embryo implantation. The Pgr Cre mouse also ablates genes in the granulosa cells of the preovulatory ovarian follicle and gonadotropes of the pituitary. To rule out an ovarian and/or pituitary cause of infertility, both Gata2 f/f and Gata2 d/d mice were subject to a superovulatory regimen of gonadotropins and the results showed that Gata2 d/d mice ovulated a similar numbers of eggs as control Gata2 f/f mice (27 +/− 3.0 vs. 23.33 +/− 4.37, respectively). Further, serum P4 levels at Day 5.5 of pregnancy were similar in both the Gata2 d/d mice and Gata2 f/f mice ( Table 1 ). Thus, the ability to ovulate and the normal serum P4 levels in combination with the fact that pituitary-specific Gata2 knock-out models are fertile ( Charles et al., 2006 ) confirmed that the Gata2 d/d infertility phenotype is not due to impairment of the hypothalamus-pituitary-ovarian axis but intrinsic to the uterus. To demonstrate that the infertile phenotype of the Gata2 d/d mice was of uterine origin, we assayed the ability of the uterus to undergo an decidual reaction in response to a regimen of E2 and P4. While Gata2 f/f mice showed typical increase in uterine weight, changes in uterine stromal cell shape and alkaline phosphatase staining in the stimulated horn compared to the unstimulated horn ( Fig. 1 F and G ), Gata2 d/d mice failed to respond and showed no increase in uterine size, weight, stroma cell morphology and alkaline phosphatase activities. ( Fig. 1 H, 1I , S1A and S1B ). In addition to these changes, the mRNA of genes known to be critical for decidualization, bone morphogenetic protein 2 ( Bmp2 ), wingless-type MMTV integration site family, member 4 ( Wnt4 ), follistatin ( Fst ), FK506 binding protein 4 ( Fkbp4 ), FK506 binding protein 5 ( Fkbp5 ) and prostaglandin-endoperoxide synthase 2 ( Ptgs2 ) were significantly attenuated in the stimulated horn of Gata2 d/d mice compared to the stimulated horn of Gata2 f/f ( Fig. S1C ). These findings collectively indicate an impaired decidualization phenotype because of Gata2 deficiency. Since the Gata2 d/d phenocopied the Pgr null mice ( Lydon et al., 1995 ), we investigated if PGR expression was affected by Gata2 deficiency. In order to control for variations of PGR levels caused by endogenous hormones, we performed PGR western blot analysis and immunostaining on uteri of ovariectomized (OVX) mice. Gata2 deficiency led to a significant decreased expression of both PGR-A and PGR-B isoforms ( Fig. 1N ) which could be seen in a reduction of PGR protein in the luminal and glandular epithelia at both baseline and in response to P4 stimulation ( Fig. 1 J-M ). These results collectively indicate that Gata2 is required for PGR expression. To determine if the reduced PGR expression in Gata2 d/d mice impaired P4 signaling, we assayed the expression of PGR target genes in OVX Gata2 d/d and Gata2 f/f uterus 6 hours following P4 (1 mg, sc) or vehicle (oil) treatment. Gata2 f/f mice showed an increase in the expression of the PGR regulated genes amphiregulin ( Areg ), cytochrome P450, family 26, subfamily A, polypeptide 1 ( Cyp26a1 ), and Indian Hedgehog ( Ihh) ( Fig. 2A-C ). In contrast, Gata2 d/d mice showed a complete loss of P4-dependent induction of Areg and Cyp26a1 ( Fig. 2 A and B ) as well as reduced Ihh expression at both baseline and in response to P4 ( Fig. 2C ). These results indicate that the P4 signaling is attenuated on PGR-dependent marker genes in Gata2 d/d mice. The global impact of Gata2 deficiency on P4 signaling was assayed by conducting microarray analysis on the uteri of OVX Gata2 f/f and Gata2 d/d mice with P4 treatment. Comparison between P4 vs. vehicle treated groups identified 1861 P4 response genes ( Fig. 2D , green box and Table S1 ) in Gata2 f/f mice, while only 139 genes responded to P4 stimulation in Gata2 d/d mice ( Fig 2D , red box and Table S2 ). Strikingly, P4 failed to regulate 1814 (97%) of P4 response genes in Gata2 d/d mice ( Fig. 2D , Venn diagram). Moreover, of the 47 P4 response genes that remained responsive to P4 stimulation in the Gata2 d/d background, many genes exhibit diminished magnitude on change of gene expression in response to P4 ( Fig. 2E ). We also identified 2461 probes that represent the Gata2 dependent transcription profile in the uterus by comparing expression arrays of Gata2 f/f and Gata2 d/d mice under P4 treatment ( Fig. 2F and Table S3 ). Based on this profile, the Ingenuity Pathway Analysis, IPA, predicted inhibition of P4 signaling (activation z-score −4.05) and activation of P4 antagonist mifepristone (activation z-score 2.09) ( Fig. 2G ). These results collectively indicate the requirement of Gata2 to sustain PGR expression for P4 signaling. Suppression of uterine E2 signaling and E2 driven epithelial proliferation is a hallmark of P4-induced preparation for subsequent embryo implantation ( Wetendorf and DeMayo, 2014 ). Compared with Gata2 f/f uteri, Gata2 d/d Day 3.5 pseudopregnancy uteri showed an increased level of phosphorylation at serine 188 on ESR1 as well as increased expression of the target Mucin 1, transmembrane (MUC1) ( Fig. S2A ) with no significant change in Esr1 mRNA levels ( Fig. S2B ). Thus, the Gata2 d/d mice exhibited higher E2 signaling activities without elevated ESR1 levels. We also observed altered proliferation of the uterine epithelia in response to heightened E2 signaling after loss of Gata2 . Normally epithelial proliferation is high at day 2.5 and diminished at day 3.5 ( Fig S2C and D ). In contrast, uterine epithelial proliferation at day 3.5 remained as high as at day 2.5 in the Gata2 d/d mice ( Fig. S2 C and D ). Taken together, these results demonstrate uninhibited E2 signaling and reduced P4 signaling as a result of Gata2 deficiency. The mechanism by which Gata2 regulates PGR expression was investigated by determining the ability of Gata2 to regulate the expression of PGR at the level of Pgr gene transcription. Pgr mRNA levels was reduced in Gata2 d/d uteri compared to controls ( Fig. 3A ). ChIP-qPCR analysis further shows enriched GATA2 occupancy at a region that consists of 3 predicted GATA binding motifs upstream and proximal to the Pgr promoter in uteri of OVX Gata2 f/f mice 1 hour post P4 treatment ( Fig. 3 B and C ). This enrichment was absent in Gata2 d/d mice and in the exon 4 negative control region devoid of GATA binding motifs ( Fig. 3 B and C ), supporting the finding of GATA2 occupancy at the Pgr locus. Results from luciferase reporter analysis in cultured epithelial cells showed that the Pgr promoter was stimulated with the full length Gata2 construct and with the amino zinc finger deletion (ΔNT) construct, but not with the carboxyl zinc finger deletion (ΔCT) construct ( Fig. 3D ). This is consistent with a previous finding that the carboxyl zinc finger domain is involved in transcriptional regulation by GATA2 ( Tong et al., 2000 ). The in vitro GATA2ΔCT mutant protein results support the in vivo functional loss of our Gata2 mutant mice in which the carboxyl zinc finger domain is deleted upon cre-mediated excision. Interestingly, deleting the amino zinc finger domain of GATA2 did not affect transactivation activity by GATA2 in the Pgr promoter ( Fig. 3D ), despite a previous report that both zinc fingers are important for suppressing the PPARγ2 promoter ( Tong et al., 2000 ). Our results demonstrate that GATA2 can enhance Pgr promoter activity with its carboxyl zinc-finger domain. Collectively, we find that Pgr is a direct downstream target of GATA2. Due to the observed interdependence between PGR and GATA2 expression in uterine P4 signaling, we hypothesized that both transcription factors are required for the regulation of target gene expression. This hypothesis was tested by conducting ChIP-Seq analysis for GATA2 on uterine chromatin isolated from OVX C57BL/6 mice treated with 1 mg P4 sc for 1 hour and immunoprecipitated for GATA2 ( Rubel et al., 2012b ). Whole uterine analysis resulted in over 22×10 6 tags mapping to unique locations in the mouse genome. A model-based analysis peak finding algorithm (MACS) ( Zhang et al., 2008 ) was utilized to normalize immunoprecipitated chromatin against uterine input generating a false discovery rate of essentially zero. This high-confidence cutoff produced 46,183 GATA2 binding intervals. Analysis for DNA motifs in the GATA2 cistrome revealed the expected GATA binding motifs as well as PGR binding motifs within the GATA2-occupying intervals ( Fig. 4A and B ). To investigate whether this PGR binding motif enrichment in GATA2 occupying sites is uterus-specific, we compared GATA2 occupancy patterns and motif enrichment between two distinct tissue types, the uterus and HPC7 hematopoietic precursor cells ( Wilson et al., 2010 ). Among 46183 uterine and 9243 HPC7 cell GATA2 occupying sites, GATA2 exhibited distinct cistrome profiles with 96.7% and 83.3% sites unique to uterus and HPC7 cells, respectively ( Fig. S3A ). This finding is similar with a previous study in which GATA2 cistromes are significantly different between precursor cells and differentiated mast cells ( Calero-Nieto et al., 2014 ). Motif analyses further revealed that the uterine GATA2 cistrome contained binding motifs enriched for nuclear receptors, such as PGR, glucocorticoid receptor (GR) and androgen receptor (AR) among others ( Table S4 ), while such enrichment was not present in the HPC7 GATA2 cistrome ( Table S4 ). Similarly, motifs of Sex Determining Region Y Box (SOX) factors are also enriched in the uterine, but not the HPC7 cell GATA2 cistrome ( Table S4 ). It is worthy to note that, SOX17 is also expressed in the uterine epithelial cells ( Rubel et al., 2012b ), where GATA2 is expressed, and functions in the process of embryo implantation ( Hirate et al., 2016 ). These findings revealed a uterine-specific GATA2 occupancy pattern that is associated with the presence of binding motifs of transcription factors, such as PGR, and SOX17 that have been known to regulate uterine function. Since PGR binding motifs are enriched in the GATA2 cistrome, we aligned the GATA2 location data with previous PGR ChIP-Seq performed for mouse uterus ( Rubel et al., 2012b ). This PGR ChIP-Seq data was also conducted on the uteri of OVX mice treated with 1 mg P4 for 1 hour. Through overlaying the GATA2- and PGR-occupying genes (defined by having at least one ChIP interval within 25 kb of annotated gene boundaries) with P4 responsive genes derived from the microarray results, we found GATA2 occupancy in 1478 (79%) and PGR occupancy in 1013 (54%) P4 response genes ( Fig. 4C ). Strikingly, GATA2 and PGR occupying sites are present together on 935 (50%) genes regulated by P4 ( Fig. 4C ). We validated the GATA2 occupancy on 13 genes (15 locations total) that are previously identified to contain PGR binding sites ( Rubel et al., 2012b ). All validated sites were within known P4 responsive genes and several of these GATA2 occupying locations directly overlapped with those of PGR occupying sites ( Fig. S3B ). In summary, these findings suggest a major regulatory mechanism of P4 response genes jointly by GATA2 and PGR. The functionality of the overlap of PGR and GATA2 intervals in the regulation of gene expression was tested by conducting luciferase reporter analysis on putative enhancers containing overlapping PGR and GATA2 binding sites from chromatin locations in two P4 target genes, Sox17 and Ihh , ( Fig. 4D ) ( Lee et al., 2006 ; Rubel et al., 2012b ). The GATA2-PGR intervals were sub-cloned into a luciferase vector containing a minimal promoter and then cotransfected with combinations of vectors expressing PGR-A, PGR-B and GATA2 to evaluate the possible regulatory preferences of these factors on these putative enhancers of P4 target genes. The results showed that luciferase activities under the control of these putative enhancers increased in response to progestin (R5020) treatment in a Pgr isoform specific manner ( Fig. 4 E and F ). No induction of these reporter genes was observed with cotransfection of the Gata2 alone. However, when Gata2 was cotransfected with the Pgr isoforms, these reporter plasmids showed a significant induction in luciferase activity in the presence of R5020 treatment ( Fig. 4 E and F ). These results are consistent with our hypothesis that PGR and GATA2 can coordinate the expression of these target genes and with the observation of GATA2 regulating PGR response genes in vivo ( Fig. 2C and S3C ). The ChIP-Seq result also identified multiple GATA2 occupying sites in the Pgr locus ( Fig. S4A ), including the proximal promoter region that contains predicted GATA binding motif and is corresponding to the Pgr promoter fragment in the luciferase construct ( Fig. S4B ). Interestingly, the Pgr proximal promoter region exhibited a high GATA2 and low PGR occupying profile ( Fig. S4B ). This occupancy pattern is consistent with the luciferase promoter assay result where GATA2 alone can enhance the Pgr promoter activity ( Fig. 3D ), which is in contrast with the GATA2/PGR co-occupying enhancers where GATA2 co-activated transcription with PGR ( Fig. 4 D-F ). Our results demonstrate that GATA2 employs two mechanisms to regulate P4 signaling through directly controlling Pgr gene transcription as well as co-regulating PGR downstream gene expression with PGR. The role of Gata2 in hormone independent gene regulation of uterine function was investigated by comparing the expression profiles of Gata2 d/d and Gata2 f/f treated only with vehicle. Microarray analysis revealed 426 genes upregulated and 535 downregulated ( Table S5 ). IPA revealed enrichment in cancer and proliferation of keratinocytes/epidermal cells ( Fig. 5A ), as evidenced by the Gata2 d/d uteri exhibiting ectopic expression of TRP63 in a layer of cells, morphologically similar to basal cells, underneath the columnar luminal epithelia ( Fig. 5D ). Since TRP63 is a molecular switch for squamous metaplasia ( Koster et al., 2004 ) associated with in utero exposure to the synthetic E2 diethylstilbestrol (DES) (Franco et al.; Goldberg and Falcone, 1999 ; Sassoon, 1999 ), we hypothesized that Gata2 deficiency may increase susceptibility in developing squamous metaplasia in response to E2 signaling. Long-term treatment of OVX mice with E2 resulted in development of focal regions of TRP63-positive cells into squamous cell metaplasia as early as 2 weeks post treatment ( Fig. 5F ), with Trp63 expression over time ( Fig. 5G and S5 ). An increase in expression of keratin genes including Krt5 and Krt15 , in Gata2 f/f mice ( Fig. 5 H and I ) was also observed. These changes were not observed in the control mice ( Fig. 5E ). In summary, our results revealed a Gata2 dependent and P4 independent mechanism in suppression of Trp63 expression and a role in squamous metaplasia. This demonstrates that Gata2 is critical for the uterine epithelium to maintain its differentiated state. The clinical relevance of our findings in mouse models was evaluated by determining whether there is an association between the GATA2 and P4 signaling in human uterine expression databases. We first identified human orthologues of Gata2 -regulated mouse genes in the presence of P4 ( Fig. 2F ). Based on these GATA2 reporter genes originally derived from the mouse ( Fig. 6A , top panel), we then determined the human GATA2 gene activities in individual endometrial tissue of 115 human samples ( GSE58144 ) ( Koot et al., 2016 ). A positive correlation between GATA2 levels and activities in human samples just as in mouse was observed ( Fig. 6A and S6A ). Moreover, in the same cohort of human samples, we found GATA2 activities are positively correlated with levels of PGR and of members of PGR signaling components PTCH1 , AREG , FKBP4 , PTGS2 and SOX17 ( Fig. 6A and S6A ). Taken together, these results suggest a conserved GATA2 regulatory network in human and mouse endometrium. Results from our studies and others revealed a regulatory network for female fertility ( Fig. 6B ) that consists of GATA2, PGR and SOX17 transcription factors ( Guimaraes-Young et al., 2016 ; Hirate et al., 2016 ; Rubel et al., 2012a ; Rubel et al., 2012b ). To investigate this network in human endometrial samples, we performed path analyses on the GSE58144 dataset using a structural equation modeling ( Xiong et al., 2004 ). First, we examined a model in which GATA2 and PGR are mutually regulated and both transcription factors jointly modulate SOX17, based on mRNA levels of each gene ( Fig. 6C ). This model significantly fits the data from GSE58144 with all proposed paths ( Fig. 6C ). The fit indices of the tested model all provided evidence of a close fit in which root mean square error of approximation (RMSEA) is less than 0.001, standardized root mean square residual (SRMR) is less than 0.001 and comparative fit index (CFI) equals to 1.0, and Tucker Lewis fit index (TLI) equals to 1.0 ( Fig S5B ). Based on results of these indices, this model is considered not rejected by the human data. Additionally, both GATA2 and PGR have significant direct effects on SOX17 ( p < 0.05). Overall, higher GATA2 and PGR levels were associated with higher SOX17 levels. Furthermore, using the GATA2 signature ( Fig. 6A ) and the PGR signature (derived from GSE39920 , Table S6 ) as molecular activities to replace GATA2 and PGR levels in the model also yielded statistically significant results ( Fig. 6D and S6B ). Collectively, these findings suggest the presence of a GATA2 - PGR - SOX17 regulatory network in the GSE58144 dataset of human endometrial tissues. Next, we tested the model of joint regulation of the P4 signaling by GATA2 and PGR . Activities of human P4 signaling in GSE58144 were generated based on a mouse P4 signature ( Fig. 2D ). Model fitting of data from GSE58144 via structural equation modeling shows statistical significance on all proposed paths ( p < 0.05) ( Fig. 6E ) while the model is considered not rejected by the human GSE58144 data based on RMSEA, SRMR, CFI and TLI indices ( Fig. S6B ). In summary, these results suggest that, in humans, GATA2 and PGR together regulate P4 signaling and form a network with SOX17 to direct a transcription program for female fertility.

Our study demonstrated that GATA2 modulates PGR signaling via regulation of Pgr transcription and through GATA2-PGR interaction in transcriptional regulation of P4 response genes. These results are consistent with a previous study that shows that binding of GATA2 with PGR is essential for the expression of a known P4 target gene Fkbp5 in mouse mammary cancer cells ( Magklara and Smith, 2009 ). Thus, in conjunction with our previous findings on regulation of Gata2 by PGR ( Rubel et al., 2012a ), we show the reciprocal regulation of Gata2 and Pgr that allows both genes to be present to regulate the transcription of key genes involved in the preparation of the mouse uterus for embryo implantation. This finds further support from the model fitting analysis in human endometrial tissues, suggesting a conservation of the molecular mechanism across species. Several studies have provided evidences for the cooperative nature of GATA2 with the androgen receptor (AR) in androgen induced gene response, while the expression of AR itself is also regulated by GATA2 ( Perez-Stable et al., 2000 ; Wu et al., 2014 ). Given that androgen response elements (AREs) contain sequence homology with that of P4 response elements (PREs), our study highlights the amazing resemblance between GATA2-PGR and GATA2-AR in regulation of gene expression. Recently, GATA2 has been reported to be down regulated in endometriosis samples in humans in Dyson et al. They reported that PGR expression failed to respond to manipulation of GATA2 levels, which is different from our observation from the in vivo mouse model. This discrepancy likely resulted from the cell type difference; we primarily observed the Gata2 - Pgr interaction in epithelial cells while Dyson et al. conducted experiments in stromal cells. Owing to the fact that OVX mice are in the hormone deprived state during which basal expression of PGR is restricted to the luminal epithelium and glands ( Fig. 1 I and K ), the epithelial derived P4 signaling is likely the primary source of acute P4 responses observed in our studies. Interestingly, as a P4 independent phenotype, ablating Gata2 in the uterus resulted in endometrial epithelial morphology alteration from simple columnar epithelium into stratified squamous epithelium, in association with increased TRP63 expression. TRP63, particularly the delta N isoform, has been suggested as a driver in the developmental switch from a simple epithelium to a squamous epidermal fate ( Romano and Sinha, 2011 ). The stratified endometrial epithelial phenotype shown here is similar to exposure to DES in fetal or neonatal mice ( Iguchi and Takasugi, 1987 ; Plapinger, 1981 ). A similar disruption of human uterine differentiation was observed when DES was mistakenly prescribed to pregnant women during 1947-1971 under the assumption to preserve pregnancy. This exposure associated with increasing reproductive tract anomalies including vaginal adenosis and endometrial cancer in their daughters ( Haney et al., 1979 ; Herbst et al., 1971 ). Due to the dependence of E2 during this process, we further demonstrated that loss of Gata2 resulted in a squamous cell metaplasia phenotype in the glandular and luminal epithelium that was marked by presence of TRP63 after chronic E2 treatment. Uterine disruption of a number of transcription factors, including Wnt signaling Wnt7a , Wnt4 , Wnt5a and Ctnnb1 ( Franco et al., 2011 ; Jeong et al., 2009 ; Mericskay et al., 2004 ; Miller and Sassoon, 1998 ), as well as Hox family member Hoxa10 , Hoxa11 , and Hoxa13 ( Benson et al., 1996 ; Zhao and Potter, 2001 ), also resulted in uterine epithelial morphological alteration. Future experiments will need to define the relationship of GATA2 with Wnt signaling in this process. Our results demonstrate that Gata2 is critical for the maintenance of normal uterine epithelia differentiation. The presence of P4- and GATA2-independent genes in the GATA2- and P4-targets, respectively, suggests involvement of additional mechanisms in controlling these 2 groups of genes. To explore potential signals that regulate expression of genes responded uniquely to P4 but not GATA2, we performed motif analysis on genes differentially expressed upon P4 stimulation in the Gata2 knockout background ( Fig. 2D , red circle). Searching for known motifs in promoter regions of these genes revealed an enrichment of AP2 and RBPJ1 binding motifs ( Table S7A ). Given that the AP2 binding motif is also enriched in the PGR occupancy sites in T47D breast cancer cell lines ( Yin et al., 2012 ), perhaps a conserved regulatory mechanism that consists of PGR and AP2 is present for a subset of PGR target genes. This result also suggests that PGR might work with multiple pioneer factors, such as GATA2 and AP2 to regulate various subsets of PGR downstream targets. On the other hand, we found a different profile of enriched motifs in promoter regions of genes downstream to GATA2 responsive genes without the need of P4 stimulation ( Tables S5 and S7B ). In this group, the binding motif for Forkhead box (FOX) proteins is enriched in the GATA2 occupying sites, which bears a resemblance of co-occupancy of GATA2 and FOXA1 in genomic loci of AR target genes in prostate cancer ( Wu et al., 2014 ). Furthermore, the enrichment of SOX (Sex Determining Region Y Box) factor binding motifs also implicates a potential interaction between GATA2 and other uterine transcription regulators such as SOX17 ( Garcia et al., 2007 ; Guimaraes-Young et al., 2016 ; Hirate et al., 2016 ). These results indicate future investigations on potential mechanisms by which GATA2 and PGR work independently with other transcription regulators to direct gene expression in the uterus. In summary, this study contributes to a more comprehensive understanding of P4 regulation through the identification of a role for the transcription factor GATA2 in uterine function. The morphological alteration of endometrial epithelium in response to Gata2 deficiency suggests that Gata2 may play a role in postnatal uterine development. Given that GATA2 activities can be modulated by small molecules ( He et al., 2014 ), fully understanding the interactions of Gata2 with the network of genes involved in the regulation of uterine function will have implications for the future treatment of female reproductive health and associated disorders.