Progesterone Receptor Binding Sites Research Articles

RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/β subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHβ subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHβ results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP+ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHβ subunit in pituitaries of women.

Transposable Elements Continuously Remodel the Regulatory Landscape, Transcriptome, and Function of Decidual Stromal Cells.

Gene expression evolution underlies the origin, divergence, and conservation of biological characters including cell-types, tissues, and organ systems. Previously we showed that large-scale gene expression changes in decidual stromal cells (DSCs) contributed to the origins of pregnancy in eutherians and the divergence of pregnancy traits in primates and that transposable elements likely contributed to these gene expression changes. Here we show that two large waves of TEs remodeled the transcriptome and regulatory landscape of DSCs, including a major wave in primates. Genes nearby TE-derived regulatory elements are among the most progesterone responsive in the genome and play essential roles in orchestrating progesterone responsiveness and the core function of decidual cells by donating progesterone receptor binding sites to the genome. We tested the regulatory abilities of 89 TE consensus sequences and found that nearly all of them acted as repressors in mammalian cells, but treatment with a histone deacetylase inhibitor unmasked latent enhancer functions. These data indicate that TEs have played an important role in the development, evolution, and function of primate DSCs and suggest a two-step model in which latent enhancer functions of TEs are unmasked after they lose primary repressor functions.

The WID-BC-index identifies women with primary poor prognostic breast cancer based on DNA methylation in cervical samples

Genetic and non-genetic factors contribute to breast cancer development. An epigenome-based signature capturing these components in easily accessible samples could identify women at risk. Here, we analyse the DNA methylome in 2,818 cervical, 357 and 227 matched buccal and blood samples respectively, and 42 breast tissue samples from women with and without breast cancer. Utilising cervical liquid-based cytology samples, we develop the DNA methylation-based Women’s risk IDentification for Breast Cancer index (WID-BC-index) that identifies women with breast cancer with an AUROC (Area Under the Receiver Operator Characteristic) of 0.84 (95% CI: 0.80–0.88) and 0.81 (95% CI: 0.76–0.86) in internal and external validation sets, respectively. CpGs at progesterone receptor binding sites hypomethylated in normal breast tissue of women with breast cancer or in BRCA mutation carriers are also hypomethylated in cervical samples of women with poor prognostic breast cancer. Our data indicate that a systemic epigenetic programming defect is highly prevalent in women who develop breast cancer. Further studies validating the WID-BC-index may enable clinical implementation for monitoring breast cancer risk.

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.

Cytostatic Activity and Ligand-Receptor Interaction Energy of the Novel Russian-Produced Gestagen Gestobutanoil and its Metabolites

With the aim of clarifying the cytostatic effect of the Russian-made gestagen gestobutanoil (17α-acetoxy-3β-hydroxy-6-methylpregna-4,6-dien-20-one butyl ester), which is a member of an original group of transformed gestagens containing an ester substituent in place of the 3-keto group, the cytostatic activities of its metabolites (17α-acetoxy-3β-hydroxy-6-methylpregna-4,6-dien-20-one (AMP-17) and megestrol acetate) and a reference agent (progesterone) were studied in uterine cervical tumor line HeLa. Complex formation by these steroids with progesterone receptor binding sites was evaluated by computer modeling (docking) by determination of the interaction energy of the partner molecules. The cytostatic activity of gestobutanoil was greater than that of reference compound and may be associated with the formation of its active metabolites. Conformations of the gestobutanoil-receptor complex with binding energies significantly different from those of complexes with progesterone, AMP-17, and megestrol acetate were demonstrated; this may be the cause of changes in the affinity of gestobutanoil for progesterone receptors and modulation of its biological activity.

Tissue-specific progesterone receptor-chromatin binding and the regulation of progesterone-dependent gene expression

Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for divergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of diverse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.

Abstract 2621A: Progesterone receptor-mediated repression of STAT5A-target genes implicated in breast cancer development

Abstract Recent clinical data suggest that progesterone and the progesterone receptor (PR) promote the development of breast cancer. PR itself is a transcription factor, and likely cooperates with other transcription factors to alter tumorigenesis in the mammary gland. ChIP-seq data from T47D breast cancer cells treated with R5020 (synthetic progesterone) showed that PR binding sites were enriched in signal transducers and activators of transcription 5A (STAT5A) gamma interferon activated sites (GAS), the genomic binding sites for STAT5A. This suggests possible adjacent or overlapping binding sites for STAT5A and PR. STAT5A is activated in a similar manner to PR: phosphorylation leads to dimerization and translocation to the nucleus, DNA binding and regulation of target genes involved in mammary gland development, inflammation, and proliferation. The clinical data surrounding the role for STAT5A in breast cancer risk and progression are mixed, and suggest that STAT5A activity in breast cancer is very context dependent, varying based on hormone receptor status. PR and STAT5A are both required for normal mammary gland development and, interestingly, when knocked out arrest at the same stage of mammary gland ductal branching. These data imply putative crosstalk between PR and STAT5A, perhaps through an interaction between these two transcription factors. Co-IP experiments in T47D cells showed an interaction between PR and STAT5A that is regulated by the addition of PR ligand and/or prolactin (STAT5A activator in the breast/mammary gland). To determine the implication of this interaction on STAT5A transcriptional activity we performed a luciferase assay. We transfected a STAT5A target gene (CISH) linked to a luciferase reporter into T47D cells stably expressing shRNA for PR and non-silencing (NS) control. Interestingly, when both ligands for STAT5A and PR were present there was repression of STAT5A transcription of the luciferase reporter compared to when STAT5A ligand was present alone. This repression was lost in cells expressing shRNA for PR. This PR dependent repression was verified in the context of chromatin looking at select STAT5A-target gene (CISH, EREG, and PTHrP), expression via qPCR; prolactin-activated expression of these genes was potently repressed in response to PR ligand treatment. Of note, one of the STAT5A-target genes shown to be repressed by PR, PTHrP (parathyroid hormone related protein), has been linked to breast cancer progression, and specifically, immune cell recruitment to mammary gland tumors. Studies are ongoing now to determine how PR-mediated repression of PTHrP may affect immune cell recruitment to mammary gland tumors models in mice. Understanding the crosstalk between PR and STAT5A, and what transcriptional programs are jointly regulated/repressed by these two transcription factors, could lead to the discovery of novel, PR-based therapies in breast cancer. Citation Format: Sean Holloran, Gloria Trinca, Christy Hagan. Progesterone receptor-mediated repression of STAT5A-target genes implicated in breast cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2621A.

Hormone-control regions mediate steroid receptor-dependent genome organization.

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers.

In silico assessment of new progesterone receptor inhibitors using molecular dynamics: a new insight into breast cancer treatment.

Nowadays, breast cancer is one of the most widespread malignancies in women, and the second leading cause of cancer death among women. The progesterone receptor (PR) is one of the treatment targets in breast cancer, and can be blocked with selective progesterone receptor modulators (SPRMs). Since administration of chemical drugs can cause serious side effects, and patients, especially those undergoing long-term treatment, can suffer harmful consequences, there is an urgent need to discover novel potent drugs. Large-scale structural diversity is a feature of natural compounds. Accordingly, in the present study, we selected a library of 20,000 natural compounds from the ZINC database, and screened them against the PR for binding affinity and efficacy. In addition, we evaluated the pharmacodynamics and ADMET properties of the compounds and performed molecular docking. Moreover, molecular dynamics (MD) simulation was carried out in order to examine the stability of the protein. In addition, principal component analysis (PCA) was performed to study the motions of the protein. Finally, the MMPBSA method was applied in order to estimate the binding free energy. Our docking results reveal that compounds ZINC00936598, ZINC00869973 and ZINC01020370 have the highest binding energy into the PR binding site, comparable with that of Levonorgestrel (positive control). Moreover, RMSD, RMSF, Rg and H-bond analysis demonstrate that the lead compounds preserve stability in complex with PR during simulation. Our PCA analysis results were in accordance with MD results and the binding free energies support the docking results. This study paves the way for discovery of novel drugs from natural sources and with optimal efficacy, targeting the PR. Graphical Abstract The binding mode of new progesterone receptor inhibitors.

5α-dihydrotestosterone reduces renal Cyp24a1 expression via suppression of progesterone receptor.

Androgens act in concert with vitamin D to influence reabsorption of calcium. However, it is unclear whether androgens directly regulate vitamin D homeostasis or control other cellular events that are related to vitamin D metabolism. To examine whether the expression of vitamin D-related genes in mouse kidney is driven by androgens or androgen-dependent effects, the androgen receptor and other sex steroid receptors were monitored in orchidectomized mice treated with 5α-dihydrotestosterone (DHT). Our results revealed that exposing orchidectomized mice to DHT inhibited the expression of progesterone receptor (Pgr) with or without estrogen receptor α expression, the latter was confirmed by ER-positive (MCF7 and T47D) or -negative (PCT) cells analysis. The loss of Pgr in turn decreased the expression of renal 24-hydroxylase via transcriptional regulation because Cyp24a1 gene has a progesterone receptor-binding site on promoter. When male kidneys preferentially hydroxylate 25-hydroxyvitamin D3 using 24-hydroxylase rather than 25-hydroxyvitamin D3-1-alpha hydroxylase, DHT suppressed the Pgr-mediated 24-hydroxylase expression, and it is important to note that DHT increased the blood 25-hydroxyvitamin D3 levels. These findings uncover an important link between androgens and vitamin D homeostasis and suggest that therapeutic modulation of Pgr may be used to treat vitamin D deficiency and related disorders.

Breast Cancer Suppression by Progesterone Receptors Is Mediated by Their Modulation of Estrogen Receptors and RNA Polymerase III.

Greater than 50% of estrogen receptor (ER)-positive breast cancers coexpress the progesterone receptor (PR), which can directly and globally modify ER action to attenuate tumor growth. However, whether this attenuation is mediated only through PR-ER interaction remains unknown. To address this question, we assessed tumor growth in ER/PR-positive patient-derived xenograft models of breast cancer, where both natural and synthetic progestins were found to antagonize the mitogenic effects of estrogens. Probing the genome-wide mechanisms by which this occurs, we documented that chronic progestin treatment blunted ER-mediated gene expression up to 2-fold at the level of mRNA transcripts. Unexpectedly, <25% of all ER DNA binding events were affected by the same treatment. The PR cistrome displayed a bimodal distribution. In one group, >50% of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR but not ER was associated with a large fraction of RNA polymerase III-transcribed tRNA genes, independent of hormone treatment. Notably, we discovered that PR physically associated with the Pol III holoenzyme. Select pre-tRNAs and mature tRNAs with PR and POLR3A colocalized at their promoters were relatively decreased in estrogen + progestin-treated tumors. Our results illuminate how PR may indirectly impede ER action by reducing the bioavailability of translational molecules needed for tumor growth. Cancer Res; 77(18); 4934-46. ©2017 AACR.

A Role for Progesterone-Regulated sFRP4 Expression in Uterine Leiomyomas.

Context:Despite progesterone’s key role in uterine smooth muscle tumorigenesis, the mechanisms by which it promotes the growth of uterine leiomyomas remain poorly understood.Objective:The aim of this study was to identify gene products mediating the effects of progesterone in uterine leiomyomas.Design:Gene expression profiling was used to identify putative progesterone-regulated genes differentially expressed in uterine leiomyomas, which were then studied in vitro.Methods:Gene expression was comprehensively profiled with the Illumina WG BeadChip (version 2.6) and analyzed with a bioinformatic algorithm that integrates known protein–protein interactions. Genomic binding sites for progesterone receptor (PR) were interrogated by chromatin immunoprecipitation—quantitative polymerase chain reaction (ChIP-qPCR). Small interfering RNA was used to study gene function in primary cell lines.Results:Our analyses identified secreted Frizzled-related protein 4 (sFRP4) as a key gene product functionally linked to PR activation whose expression was 2.6 times higher in leiomyomas than myometrium (n = 26, P < 0.01) and 2.5 times higher during the proliferative phase of the menstrual cycle (n = 26, P < 0.01). Direct binding between PR and sFRP4 promoter was observed by ChIP-qPCR. Robust overexpression of sFRP4 was also observed in primary cultures derived from leiomyoma. Progesterone preferentially inhibited sFRP4 expression and secretion in leiomyoma cultures in a dose-dependent manner sensitized by estradiol. Knockdown of sFRP4 inhibited proliferation and apoptosis in primary cultures of both myometrium and leiomyoma.Conclusions:Overexpression of sFRP4 is a robust, progesterone-regulated feature of leiomyomas that increases smooth muscle proliferation. More work is needed to elucidate how progesterone’s ability to modulate sFRP4 expression contributes to uterine smooth muscle tumorigenesis.

Bioinformatic analysis of cis-regulatory interactions between progesterone and estrogen receptors in breast cancer.

Chromatin factors interact with each other in a cell and sequence-specific manner in order to regulate transcription and a wealth of publically available datasets exists describing the genomic locations of these interactions. Our recently published BiSA (Binding Sites Analyser) database contains transcription factor binding locations and epigenetic modifications collected from published studies and provides tools to analyse stored and imported data. Using BiSA we investigated the overlapping cis-regulatory role of estrogen receptor alpha (ERα) and progesterone receptor (PR) in the T-47D breast cancer cell line. We found that ERα binding sites overlap with a subset of PR binding sites. To investigate further, we re-analysed raw data to remove any biases introduced by the use of distinct tools in the original publications. We identified 22,152 PR and 18,560 ERα binding sites (<5% false discovery rate) with 4,358 overlapping regions among the two datasets. BiSA statistical analysis revealed a non-significant overall overlap correlation between the two factors, suggesting that ERα and PR are not partner factors and do not require each other for binding to occur. However, Monte Carlo simulation by Binary Interval Search (BITS), Relevant Distance, Absolute Distance, Jaccard and Projection tests by Genometricorr revealed a statistically significant spatial correlation of binding regions on chromosome between the two factors. Motif analysis revealed that the shared binding regions were enriched with binding motifs for ERα, PR and a number of other transcription and pioneer factors. Some of these factors are known to co-locate with ERα and PR binding. Therefore spatially close proximity of ERα binding sites with PR binding sites suggests that ERα and PR, in general function independently at the molecular level, but that their activities converge on a specific subset of transcriptional targets.

Regulation of the gene encoding human thrombin-activatable fibrinolysis inhibitor by estrogen and progesterone

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-carboxypeptidase B-like pro-enzyme that upon activation attenuates fibrinolysis and inflammatory processes. There is a large inter-individual variability in plasma TAFI levels within the population which is primarily due to epigenetic factors. Novel associations between plasma TAFI levels and sex steroids have triggered interest in determining the role of TAFI as a mediator of the cardioprotective effects of estrogens and progestins, or as a mediator of the increased thrombotic risk that accompanies use of oral contraceptives or hormone replacement therapy (HRT). In this study, we measured the effect of sex steroids on hepatic expression of CPB2, the human gene encoding TAFI. Using human hepatocellular carcinoma cells cultured in the presence of progesterone and 17β-estradiol, we demonstrated that the level of TAFI protein is decreased by those sex steroids. These changes in protein expression were paralleled by decreases in CPB2 mRNA abundance and promoter activity. We did not find evidence of estrogen or progesterone receptor binding sites in the CPB2 promoter region, suggesting that the genomic effects of progesterone and 17β-estradiol are mediated indirectly, without receptor binding to the CPB2 promoter. Indeed, we found that the effect of estrogen was independent of the estrogen receptor and was mediated through a novel signaling pathway dependent on phosphatidylinositol 3-kinase and Akt that did not involve GPR30. Our findings provide the molecular explanation for the ability of female sex steroids to decrease plasma TAFI concentrations.

Progesterone receptor induces bcl-x expression through intragenic binding sites favoring RNA polymerase II elongation

Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3′-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes.

Activation of mitogen- and stress-activated kinase 1 is required for proliferation of breast cancer cells in response to estrogens or progestins

Growth of breast cancers is often dependent on ovarian steroid hormones making the tumors responsive to antagonists of hormone receptors. However, eventually the tumors become hormone independent, raising the need to identify downstream targets for the inhibition of tumor growth. One possibility is to focus on the signaling mechanisms used by ovarian steroid hormones to induce breast cancer cell proliferation. Here we report that the mitogen- and stress-activated kinase 1 (MSK1) could be a potential druggable target. Using the breast cancer cell line T47D, we show that estrogens (E2) and progestins activate MSK1, which forms a complex with the corresponding hormone receptor. Inhibition of MSK1 activity with H89 or its depletion by MSK1 short hairpin RNAs (shRNAs) specifically abrogates cell proliferation in response to E2 or progestins without affecting serum-induced cell proliferation. MSK1 activity is required for the transition from the G1- to the S-phase of the cell cycle and inhibition of MSK1 compromises both estradiol- and progestin-dependent induction of cell cycle genes. ChIP-seq experiments identified binding of MSK1 to progesterone receptor-binding sites associated with hormone-responsive genes. MSK1 recruitment to epigenetically defined enhancer regions supports the need of MSK1 as a chromatin remodeler in hormone-dependent regulation of gene transcription. In agreement with this interpretation, expression of a histone H3 mutated at S10 eliminates the hormonal effect on cell proliferation and on induction of relevant target genes. Finally, we show that E2- or progestin-dependent growth of T47D cells xenografted in immunodefficient mice is inhibited by depletion of MSK1, indicating that our findings are not restricted to cultured cells, and that MSK1 plays an important role for hormone-dependent breast cancer growth in a more physiological context.

Abstract 3567: Estrogen signaling promotes effective progesterone receptor engagement with DNA in breast cancer cells.

Abstract Estrogens and progesterone (PROG) are key mediators of breast cancer aetiology and progression. The predominant actions of these hormones in the breast are mediated via their respective cognate steroid receptors, estrogen receptor alpha (ERα) and progesterone receptor (PGR). As ERα upregulates PGR expression, both are currently measured in clinical breast cancer samples to determine the utility of anti-estrogen therapy. Although the molecular actions of estrogens have been extensively studied, the role of PROG and PGR need further elucidation, particularly in the context of activated ERα signaling. Here, we treated ZR75-1 cells with 17β-estradiol (E2) alone or in combination with PROG and assessed global transcriptional responses by microarray expression profiling and receptor binding profiles by chromatin immunoprecipitation-sequencing (ChIP-seq). PROG treatment alone significantly altered the regulation of only seven genes, whereas long term E2 treatment increased the PROG transcriptional response altering 785 genes (p value ≤ 0.05), and regulated pathways involved in ErbB signalling and cell proliferation. Consistent with these findings, ChIP-seq identified 10 times more PGR binding sites following long term E2 treatment (4566 at equivalent peak threshold) compared with PROG treatment alone. Candidate PGR ChIP validated these findings at 100% (12/12) sites tested. The long term E2-treated PGR cistrome was highly conserved amongst species, and enriched for progesterone receptor response elements (PREs) and FOXA1 binding sites. E2 enhancement of PGR binding could be abrogated by long term (72h) but not short term (4h) tamoxifen co-treatment. In T-47D cells, long term E2 treatment was not required for efficient interaction of PGR with DNA and FOXA1 silencing resulted in decreased PGR DNA occupancy at several sites. These data suggests that the combination of E2 and PROG regulated a distinct transcriptional response in breast cancer cells, perhaps due to the dependence of PGR on E2 signaling for maintenance of cellular PGR levels, and via the receptors sharing mutual collaborators and coregulators, such as FOXA1. ERα and PGR collectively shape a mitogenic response in breast cancer cells that is dependent on the combined presence of E2 and PROG. Citation Format: Erin E. Swinstead, Andrew P. Trotta, Eric Smith, Phulwinder K. Grover, Eleanor F. Need, Grant Buchanan. Estrogen signaling promotes effective progesterone receptor engagement with DNA in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3567. doi:10.1158/1538-7445.AM2013-3567

Nucleosome-Driven Transcription Factor Binding and Gene Regulation

Elucidating the global function of a transcription factor implies the identification of its target genes and genomic binding sites. The role of chromatin in this context is unclear, but the dominant view is that factors bind preferentially to nucleosome-depleted regions identified as DNaseI-hypersensitive sites (DHS). Here we show by ChIP, MNase, and DNaseI assays followed by deep sequencing that the progesterone receptor (PR) requires nucleosomes for optimal binding and function. In breast cancer cells treated with progestins, we identified 25,000 PR binding sites (PRbs). The majority of these sites encompassed several copies of the hexanucleotide TGTYCY, which is highly abundant in the genome. We found that functional PRbs accumulate around progesterone-induced genes, mainly in enhancers. Most of these sites overlap with DHS but exhibit high nucleosome occupancy. Progestin stimulation results in remodeling of these nucleosomes with displacement of histones H1 and H2A/H2B dimers. Our results strongly suggest that nucleosomes are crucial for PR binding and hormonal gene regulation.

Non-Overlapping Progesterone Receptor Cistromes Contribute to Cell-Specific Transcriptional Outcomes

The transcriptional effects of the ovarian hormone progesterone are pleiotropic, and binding to DNA of the nuclear progesterone receptor (PR), a ligand-activated transcription factor, results in diverse outcomes in a range of target tissues. To determine whether distinct patterns of genomic interaction of PR contribute to the cell specificity of the PR transcriptome, we have compared the genomic binding sites for PR in breast cancer cells and immortalized normal breast cells. PR binding was correlated with transcriptional outcome in both cell lines, with 60% of progestin-regulated genes associated with one or more PR binding regions. There was a remarkably low overlap between the PR cistromes of the two cell lines, and a similarly low overlap in transcriptional targets. A conserved PR binding element was identified in PR binding regions from both cell lines, but there were distinct patterns of enrichment of known cofactor binding motifs, with FOXA1 sites over-represented in breast cancer cell binding regions and NF1 and AP-1 motifs uniquely enriched in the immortalized normal line. Downstream analyses suggested that differential cofactor availability may generate these distinct PR cistromes, indicating that cofactor levels may modulate PR specificity. Taken together these data suggest that cell-specificity of PR binding is determined by the coordinated effects of key binding cofactors.

Epithelial progesterone receptor exhibits pleiotropic roles in uterine development and function

The ovarian steroid progesterone, acting through the progesterone receptor (PR), coordinates endometrial epithelial-stromal cell communication, which is critical for its development and function. PR expression in these cellular compartments is under tight temporal and endocrine control. Although ex vivo studies demonstrated the importance of stromal PR expression, they failed to show a role for epithelial PR in uterine function. Here, the in vivo role of PR in the uterine epithelium is defined using floxed PR (PR(f/f)) mice crossed to Wnt7a-Cre mice. Progesterone was unable to stimulate the expression of its epithelial target genes, including Ihh, in the Wnt7a-Cre(+)PR(f/-) mice. Analysis was conducted on Ihh to determine whether PR directly regulates epithelial gene transcription. ChIP-on-chip analysis identified PR binding sites in the 5'-flanking region of Ihh. Cotransfection of the proximal Ihh promoter with PR demonstrated that PR directly regulates Ihh transcription. Female Wnt7a-Cre(+)PR(f/-) mice are infertile due to defects in embryo attachment, stromal cell decidualization, and the inability to cease estrogen-induced epithelial cell proliferation. Finally, progesterone was unable to inhibit neonatal endometrial glandular development in Wnt7a-Cre(+)PR(f/-) mice. Thus, epithelial PR is necessary for the regulation of progesterone epithelial target gene expression, as well as uterine function and development.