Progesterone Response Element Research Articles

Measuring Transcription Factor–Binding Site Turnover: A Maximum Likelihood Approach Using Phylogenies

A major mode of gene expression evolution is based on changes in cis-regulatory elements (CREs) whose function critically depends on the presence of transcription factor–binding sites (TFBS). Because CREs experience extensive TFBS turnover even with conserved function, alignment-based studies of CRE sequence evolution are limited to very closely related species. Here, we propose an alternative approach based on a stochastic model of TFBS turnover. We implemented a maximum likelihood model that permits variable turnover rates in different parts of the species tree. This model can be used to detect changes in turnover rate as a proxy for differences in the selective pressures acting on TFBS in different clades. We applied this method to five TFBS in the fungi methionine biosynthesis pathway and three TFBS in the HoxA clusters of vertebrates. We find that the estimated turnover rate is generally high, with half-life ranging between ∼5 and 150 My and a mode around tens of millions of years. This rate is consistent with the finding that even functionally conserved enhancers can show very low sequence similarity. We also detect statistically significant differences in the equilibrium densities of estrogen- and progesterone-response elements in the HoxA clusters between mammal and nonmammal vertebrates. Even more extreme clade-specific differences were found in the fungal data. We conclude that stochastic models of TFBS turnover enable the detection of shifts in the selective pressures acting on CREs in different organisms.The analysis tool, called CRETO (Cis-Regulatory Element Turn-Over) can be downloaded from http://www.bioinf.uni-leipzig.de/Software/creto/.

Detection of potential (anti)progestagenic endocrine disruptors using a recombinant human progesterone receptor binding and transactivation assay

The present work describes the identification of (anti)progestin endocrine disrupting chemicals (EDC) using a two step screening system. In the first step a competitive binding assay was developed using recombinant human progesterone receptor (hPR). The tested chemicals were of various classes like insecticides, their metabolites, industrial chemicals and waste water treatment plant (WWTP) effluents. All the tested chemicals demonstrated a high affinity binding for hPR. The average IC50 values of the test chemicals were within the range of 1-25microM. In the second step of screening, a mammalian cell-based hPR transactivation assay was developed where HEK 293 cells were co-transfected with hPR and luciferase reporter gene under the control of progesterone-response element. Stimulation of the cells with progesterone resulted in about 25-fold up regulation of luciferase activity, with EC50 value of 4nM. Potent anti-progesterone, RU486, significantly inhibited progesterone-induced transactivation and non-progestagenic steroids failed to transactivate hPR till 1microM concentrations. The chemicals showing high binding affinities in competitive binding assays were then tested in transactivation assay and all of them were found to be anti-progestative except WWTP effluents. Transactivation assays using extracted water samples from five different WWTP effluents showed that it was rich in progestative compounds. The levels of induction caused by these effluents were in the range of 15-25% of induction by progesterone and they represented about 6ng/l equivalent progesterone activities. In conclusion, we demonstrated that this two step assay provides an efficient screening tool for the detection of (anti)progestative EDC in various samples.

Progesterone regulation of RUSH/SMARCA3/HLTF includes DNA looping

RUSH/SMARCA3 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3) is capable of sequence-selective DNA binding and ATP-dependent DNA unwinding. In rabbit uterine epithelial cells, RUSH-1alpha (113 kDa) is the progesterone-dependent splice variant and RUSH-1beta (95 kDa) is the oestrogen-dependent splice variant. Rabbit RUSH/SMARCA3 mRNA is primarily regulated at the proximal promoter (-162/+90) via a PRE (progesterone-response element) half-site/overlapping Y-box domain (-38/-26) and two Sp (specificity protein) 3 sites centred at -128 and -58. We investigated hormone regulation by exploring binding of transcription factors to a putative RUSH/SMARCA3 site (-616/-611) and the distal Sp3 (-131/-126) site. In response to progesterone, RUSH-1alpha binds the RUSH site and the Sp3 site becomes a functional binding site for Egr-1 (early growth-response gene product 1)/Sp (specificity protein)1/3/MAZ (Myc-associated zinc-finger protein)/MZF1 (myeloid zinc finger 1)/c-Rel. TransSignal TF-TF Interaction Arrays, supershift assays and ChIP (chromatin immunoprecipitation) analyses confirmed strong physical interactions between RUSH and Egr-1/c-Rel. Higher-order long-range interactions between RUSH and the Egr-1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown with 3C (chromosome conformation capture) assays. Transient transfection assays with mutant constructs showed the co-operative interaction between RUSH and Egr-1 mediates repression by c-Rel. Thus DNA-bound RUSH/SMARCA3 communicates with its own proximal promoter by looping the intervening DNA. Moreover, progesterone-dependent DNA looping is an adjunct to progesterone induction of the RUSH/SMARCA3 gene because the availability of RUSH isoforms and relevant binding partners is progesterone-regulated.

Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE binding, whereas the DNA architectural properties of HMGB are dispensable. Specific PRE DNA inhibited HMGB binding to the CTE, indicating that DNA and HMGB–CTE interactions are mutually exclusive. Exogenous CTE peptide increased PR-binding affinity for PRE as did deletion of the CTE. In a PR-binding site selection assay, A/T sequences flanking the PRE were enriched by HMGB, indicating that PR DNA-binding specificity is also altered by HMGB. We conclude that a transient HMGB–CTE interaction alters a repressive conformation of the flexible CTE enabling it to bind to preferred sequences flanking the PRE.

Progesterone-Mediated Regulation of Catechol-O-Methyl Transferase Expression in Endometrial Cancer Cells

The effects of estrogen and progesterone on the expression of estrogen-metabolizing enzymes such as catechol-O-methyl transferase (COMT) are not known. COMT converts genotoxic catecholestrogens to anticarcinogenic methoxyestrogens in the endometrium. The aim of this study is to investigate the effect of progesterone on COMT expression in well-differentiated endometrial cancer cells. The wild-type Ishikawa cell line as well as progesterone receptor A- or progesterone receptor B-transfected Ishikawa cells were used for in vitro studies. The regulation of COMT expression by progesterone was studied using Western blots, Hoechst dye DNA proliferation studies, and wild-type and/or site-directed mutagenesis of COMT promoter 1-luciferase reporter gene. Progesterone upregulated COMT protein expression in Ishikawa cells through progesterone receptor A isoform. COMT promoter activity was differentially regulated by the 3 half-site progesterone response elements in the COMT promoter. High doses of 2-ME2 inhibited Ishikawa cell proliferation. These data suggest that COMT expression is hormonally regulated in well-differentiated human endometrial cancer cells. COMT regulation and 2-ME2 production in the endometrium may affect endometrial carcinogenesis.

Thermodynamic Dissection of Progesterone Receptor Interactions at the Mouse Mammary Tumor Virus Promoter: Monomer Binding and Strong Cooperativity Dominate the Assembly Reaction

Progesterone receptors (PRs) play critical roles in eukaryotic gene regulation, yet the mechanisms by which they assemble at their promoters are poorly understood. One of the few promoters amenable to analysis is the mouse mammary tumor virus gene regulatory sequence. Embedded within this sequence are four progesterone response elements (PREs) corresponding to a palindromic PRE and three half-site PREs. Early mutational studies indicated that the presence of all four sites generated a synergistic and strong transcriptional response. However, DNA binding analyses suggested that receptor assembly at the promoter occurred in the absence of significant cooperativity. Taken together, the results indicated that cooperative interactions among PREs could not account for the observed functional synergy. More broadly, the studies raised the question of whether cooperativity was a common feature of PR-mediated gene regulation. As a step toward obtaining a quantitative and, thus, predictive understanding of receptor function, we have carried out a thermodynamic dissection of PR A-isoform interactions at the mouse mammary tumor virus promoter. Utilizing analytical ultracentrifugation and quantitative footprinting, we have resolved the microscopic energetics of PR A-isoform binding, including cooperativity terms. Our results reveal a model contrary to that inferred from previous biochemical investigations. Specifically, the binding unit at a half-site is not a receptor dimer but is instead a monomer; monomers bound at half-sites are capable of significant pairwise cooperative interactions; occupancy of all three half-sites is required to cooperatively engage the palindromic-bound dimer; and finally, large unfavorable forces accompany assembly. Overall, monomer binding accounts for the majority of the intrinsic binding energetics and cooperativity contributes an approximately 1000-fold increase in receptor–promoter stability. Finally, the partitioning of cooperativity suggests a framework for interpreting in vivo transcriptional synergy. These results highlight the insight available from rigorous analysis and demonstrate that receptor–promoter interactions are considerably more complex than typically envisioned.

Progesterone Receptor Rapid Signaling Mediates Serine 345 Phosphorylation and Tethering to Specificity Protein 1 Transcription Factors

Human progesterone receptors (PR) rapidly activate cytosolic signaling pathways, in addition to their classical function as ligand-activated transcription factors. Using ER+/PR-B+ T47D breast cancer cells, we probed the role of progestin-stimulated rapid PR signaling in the transcriptional regulation of target genes involved in breast cancer cell proliferation. Epidermal growth factor receptor (EGFR) was rapidly activated after a 10-min treatment with R5020. Progestin induced EGFR-, c-Src-, and MAPK-dependent phosphorylation of PR-B on the MAPK consensus site, Ser345. Ser345-phosphorylated PR-B receptors strongly associated with specificity protein 1 (Sp1) transcription factors to regulate PR cell cycle (p21) and growth-promoting (EGFR) target genes whose promoters lack canonical progesterone response element sequences. Inhibitors of EGFR, c-Src, or MAPK activities blocked PR tethering to Sp1 and progestin-stimulated S-phase entry. Mutant PR-B receptors defective for c-Src binding (mPro) were not phosphorylated on Ser345 in response to progestin and failed to interact with Sp1. Hormone-induced complexes containing Sp1 and wild-type PR-B, but not S345A or mPro PR-B, were recruited to Sp1 sites within the endogenous p21 promoter. Progestin-induced S-phase entry was attenuated in T47D cells containing wild-type PR-B and treated with EGFR, c-Src, or MAPK kinase inhibitors or in T47D cells stably expressing mPro or mutant DNA-binding domain PR-B. In sum, rapid progestin-activated PR signaling leads to PR Ser345 phosphorylation and tethering to Sp1. These events are critical for progestin-stimulated regulation of Sp1 target genes and breast cancer cell proliferation. Our data demonstrate the therapeutic potential for PR-targeted breast cancer treatment by exploiting multiple nodes along the PR signaling pathway, including PR-B, EGFR, c-Src, MAPK, or Sp1.

Gene expression profiling of human peri-implantation endometria between natural and stimulated cycles

To investigate the effect of high serum E(2) levels in gonadotropin-stimulated cycles (hCG+7) on the gene expression patterns of human endometrium compared with natural cycles on the seventh day of LH surge (LH+7) and elucidate the underlying molecular changes that may be related to endometrial receptivity. Observational study. University Hospital. Infertile patients with normal menstrual cycles undergoing IVF treatment. Gonadotropin stimulation and endometrial biopsy. Gene expression by microarray and quantitative polymerase chain reaction (qPCR). Endometrial samples from natural (n = 5) and stimulated (n = 8) cycles were collected. Patients in the stimulated cycles were classified as moderate (n = 4) or excessive (n = 4) responders if their serum E(2) levels on the day of administration of hCG were <or=20,000 pmol/L or >20,000 pmol/L, respectively. The RNA transcripts were profiled by Affymetrix HG-U133A microarray. Clustering and principal component analysis demonstrated a significant difference (>or=2-fold) in the expression patterns of 411 genes among the three groups. Putative estrogen response elements or progesterone response elements were identified in the promoter regions of 49 differentially expressed genes of diverse biologic functions. The qPCR confirmed the microarray result in 47 endometrial samples. High serum E(2) and/or progesterone modulate the gene expression profiles of human endometrium and may affect endometrial receptivity.

Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay

The present study was aimed to develop a sensitive, fast and user friendly progesterone receptor transactivation assay using recombinant yeast cells, Saccharomyces cerevisiae, modified to express human progesterone receptor (PR) and progesterone response element (PRE) driving the expression of green fluorescent protein. Stimulation of cells with increasing concentrations of progesterone resulted in significant elevation in fluorescence activity, with the minimum effective dose of progesterone being 0.1nM. RU486, significantly inhibited progesterone induced transactivation and non-progesterogenic steroids failed to transactivate PR till 10μM concentrations. About 7 different chemicals (mostly pesticides or their metabolites) like DDT and its metabolites, nonylphenol, endosulfan were screened in this assay system for their role in transactivation and they were all found to be anti-progestative and IC50 values within the range of 3–20μM. Further, the assay was used to analyze the endocrine disrupting activity of extracted water samples from leather industries known for their high content of various chemicals and it was found to be rich in anti-progestative compounds. It resulted in about 30% reduction in transactivation. In conclusion, we demonstrated that this yeast based bioassay provides a rapid and robust assay for high throughput screening of (anti)progestative compounds from various sources.

Progesterone Receptor (PR) Isoforms PRA and PRB Differentially Regulate Expression of the Breast Cancer Resistance Protein in Human Placental Choriocarcinoma BeWo Cells

Breast cancer resistance protein (BCRP) plays a significant role in drug disposition and in conferring multidrug resistance in cancer cells. Previous studies have shown that steroid hormones such as 17beta-estradiol and progesterone can affect BCRP expression in cancer cells. In this study, we investigated the molecular mechanism by which BCRP expression in human placental choriocarcinoma BeWo cells is regulated by progesterone. Transfection of the progesterone receptor (PR) isoforms PRA and PRB resulted in a similarly increased expression of PRA and PRB, respectively. However, progesterone significantly increased BCRP expression and activity only in PRB-transfected cells. This stimulatory effect of progesterone was abrogated by the PR antagonist mifepristone (RU-486). Consistently, transcriptional activity of the BCRP promoter was induced 2- to 6-fold by 10(-8) to 10(-5) M progesterone in PRB-transfected cells. Progesterone had little effect on BCRP expression and activity and transcriptional activity of the BCRP promoter in PRA-transfected cells; however, cotransfection of PRA and PRB significantly decreased the progesterone-response compared with that in cells transfected with only PRB. Mutations in a novel progesterone response element (PRE) identified between -243 to -115 bp of the BCRP promoter region significantly attenuated the progesterone-response in PRB-transfected cells, and deletion of the PRE nearly completely abrogated the progesterone effect. Specific binding of both PRA and PRB to the BCRP promoter through the identified PRE was confirmed using the electrophoretic mobility shift assay. Collectively, progesterone induces BCRP expression in BeWo cells via PRB but not PRA. PRA represses the PRB activity. Thus, PRA and PRB differentially regulate BCRP expression in BeWo cells.

Upregulation of protein S by progestins.

Users of progestin-only contraceptives have raised protein S (PS) levels compared with baseline. This contrasts with the reduction in PS levels observed in users of combined oral contraceptives, which contain both a progestin and an estrogen. To determine the effect of progesterone and other progestin isoforms on the expression of PS and to describe the mechanism involved. Promoter activity of the PROS1 gene that encodes PS was assessed in vitro using breast and liver carcinoma cell lines grown in the presence of various progestins, with and without the addition of excess progesterone receptors. An electromobility shift assay (EMSA) was also performed to identify the progesterone receptor binding element. PROS1 transcriptional levels were directly upregulated by 25% by progesterone via a mechanism that was progesterone receptor isoform B (PR-B)-dependent. The process was blocked by the progesterone receptor modulator RU486. Results for the EMSA demonstrated that a probe comprising nucleotides -397 to -417 of the PROS1 promoter bound to ligand-activated PR-B, suggesting that the domain is a progesterone response element (PRE). The type of progestin isoform greatly influenced the level of PROS1 promoter upregulation, with medroxyprogesterone able to stimulate a > 2-fold stronger response compared with progesterone. The PROS1 promoter is responsive to progesterone and other progestins via a mechanism involving PR-B interacting with a PRE. The type of progestin is important as some elicit stronger upregulatory effects than others, which may influence the choice of progestin used for hormonal contraception by PS-deficient individuals.

Surprises with antiprogestins: novel mechanisms of progesterone receptor action.

When hormone antagonists have inappropriate agonist-like effects, the clinical consequences are grave. We describe novel molecular mechanisms by which antiprogestin-occupied progesterone receptors behave like agonists. These mechanisms include agonist-like transcriptional effects that do not require receptor binding to DNA at progesterone response elements, or that result from cross-talk between progesterone receptors and other signalling pathways. We discuss the complex structural organization of progesterone receptors, and demonstrate that the B receptor isoform has a unique third activation domain that may confer agonist-like properties in the presence of antiprogestins, whereas the A receptor isoform is a dominant-negative inhibitor. We argue that these novel mechanisms play a role in the apparent hormone resistance of breast cancers and the variable tissue-specific responses to antagonists.

Progesterone Receptor Regulates Bcl-2 Gene Expression through Direct Binding to Its Promoter Region in Uterine Leiomyoma Cells

Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.

Some Motifs Were Important for Myostatin Transcriptional Regulation in Sheep (Ovis aries)

Many motifs along the 1.2 kb myostatin promoter (MSTNpro) in sheep have been found by the MatInspecter program in our recent study. To further verify the role of the motifs and better understand the transcriptional regulation mechanism of the myostatin gene in sheep, the reporter gene EGFP (enhanced green fluorescent protein) was selected and the wild-type (W) vector MSTNPro(W)-EGFP or motif-mutational (M) vector MSTNPro(M)-EGFP were constructed. The transcriptional regulation activities were analyzed by detecting the fluorescence strength of EGFP in C2C12 myoblasts transfected with the vectors. The results showed that E-box (E) 3, E4, E5 and E7, particularly E3, E5 and E7, had important effects on the activity of the 1.2 kb sheep myostatin promoter. In addition, we also detected several other important motifs such as MTBF (muscle-specific Mt binding factor), MEF2 (myocyte enhancer factor 2), GRE (glucocorticoid response elements) and PRE (progesterone response elements) along the sheep myostatin promoter by the mutational analysis.

Identification of 9 uterine genes that are regulated during mouse pregnancy and exhibit abnormal levels in the cyclooxygenase-1 knockout mouse

BackgroundPreterm birth is the leading cause of all infant mortality. In 2004, 12.5% of all births were preterm. In order to understand preterm labor, we must first understand normal labor. Since many of the myometrial changes that occur during pregnancy are similar in mice and humans and mouse gestation is short, we have studied the uterine genes that change in the mouse during pregnancy. Here, we used microarray analysis to identify uterine genes in the gravid mouse that are differentially regulated in the cyclooxygenase-1 knockout mouse model of delayed parturition.MethodsGestational d18.0 uteri (n = 4) were collected from pregnant wild-type and cyclooxygenase-1 knockout mice. Part of the uterus was used for frozen sections and RNA was isolated from the remainder. Microarray analysis was performed at the Indiana University School of Medicine Genomic Core and analyzed using the Microarray Data Portal. Northern analysis was performed to confirm microarray data and the genes localized in the gravid uterus by in situ hybridization.ResultsWe identified 277 genes that are abnormally expressed in the gravid d18.0 cyclooxygenase-1 knockout mouse. Nine of these genes are also regulated in the normal murine uterus during the last half of gestation. Many of these genes are involved in the immune response, consistent with an important role of the immune system in parturition. Expression of 4 of these genes; arginase I, IgJ, Tnfrsf9 and troponin; was confirmed by Northern analysis to be mis-regulated during pregnancy in the knockout mouse. In situ hybridization of these genes demonstrated a similar location in the gravid wild-type and Cox-1 knockout mouse uteri.ConclusionTo our knowledge, this is the first work to demonstrate the uterine location of these 4 genes in the mouse during late pregnancy. There are several putative transcription factor binding sites that are shared by many of the 9 genes identified here including; estrogen and progesterone response elements and Ets binding sites. In summary, this work identifies 9 uterine murine genes that may play a role in parturition. The function of these genes is consistent with an important role of the immune system in parturition.

Rational Design, Synthesis, and Biological Evaluation of Progesterone-Modified MRI Contrast Agents

A series of contrast agents for magnetic resonance imaging (MRI) aimed at noninvasively determining the hormone receptor status of cancer in vitro was developed. These MRI contrast agents were prepared by conjugating progesterone to clinically used Gd(III) chelates. These agents exhibited higher progesterone receptor binding affinities in the nanomolar range and intracellular accumulation. High logP values of the modified compounds suggested that the lipophilicity of the steroid conjugates may have contributed to membrane permeability. Synchrotron radiation X-ray fluorescence microscopy and magnetic resonance images revealed that the synthesized conjugates showed the greatest cellular accumulation and significant increase in relaxivity in vitro compared to the previously developed steroid-modified agent. Transcriptional assays using the progesterone response element linked to luciferase indicated that the contrast agents entered the cell, interacted with the biological target, and drove specific progesterone-mediated transcription.

Progesterone regulates catechol- O-methyl transferase gene expression in breast cancer cells: Distinct effect of progesterone receptor isoforms

There is strong evidence that catechol- O-methyl transferase (COMT) protects breast cells against estrogen-induced cancer by detoxifying catecholestrogens, the carcinogenic estrogen metabolites. COMT gene expression is controlled by two promoters – a proximal promoter (COMTP1) and a distal promoter (COMTP2) – that regulate the expression of soluble (S-COMT) and membrane-bound (MB-COMT) isoforms, respectively. We investigated the transcriptional regulation of the COMT gene by progesterone/progesterone receptors in breast cancer cells. Our results indicated that progesterone (P4) downregulates COMT gene expression in breast cancer cell lines. In addition, the COMTP1 and COMTP2 harbor several progesterone response elements (PREs). Electrophoretic mobility shift assay (EMSA) indicated that nuclear extracts of T47D cells bind to the identified PREs in COMTP1. Site-directed mutagenesis of PREs in COMTP1 not only reversed the P4-induced inhibition of COMTP1, but also increased its basal activity. The two progesterone receptor isoforms, PR-A and PR-B, were found to have opposite effects on the regulation of P4 in COMT expression; PR-A is associated with P4-induced upregulation of COMT, while PR-B is associated with P4-induced downregulation of COMT. In summary, our data demonstrated that P4 downregulates the COMT gene expression through multiple PREs in the COMT promoters and that different progesterone receptor isoforms have distinctive effects on COMT gene expression.

The Role of Extranuclear Signaling Actions of Progesterone Receptor in Mediating Progesterone Regulation of Gene Expression and the Cell Cycle

Human progesterone receptor (PR) contains a motif that interacts with the SH3 domain of Src and mediates rapid activation of Src and downstream MAPK (Erk-1/-2) without relying on the transcriptional activity of the receptor. Here we investigated the role and intracellular location of this nontranscriptional activity of PR. Progestin activation of Src/MAPK occurred outside the nucleus with the B isoform of PR that was distributed between the cytoplasm and nucleus, but not with PR-A that was predominantly nuclear. Breast cancer cells stably expressing wild-type PR-B or PR-B with disrupting point mutations in the SH3 domain binding motif (PR-BDeltaSH3) that do not affect the transcriptional activity of PR, were compared for effects of progestin on endogenous target gene expression and cell proliferation. Progestin induction of the cyclin D1 gene, which lacks a progesterone response element, was dependent on PR activation of the Src/MAPK pathway, whereas induction of the Sgk (serum and glucocorticoid regulated kinase) gene that contains a functional progesterone response element was unaffected by mutations that interfere with PR activation of Src. Progestin induction of cell cycle progression was also abrogated in cells expressing PR-BDeltaSH3, and no effect of progestin on cyclin D1 expression and cell cycle was observed in the presence of PR-A. These results highlight the importance of PR activation of the Src/MAPK signaling pathway for progesterone-induced transcription of select target genes and cell cycle progression.

Establishing a statistic model for recognition of steroid hormone response elements

Identification of hormone response elements (HREs) is essential for understanding the mechanism of hormone-regulated gene expression. To date, there has been a lack of effective bioinformatics tools for recognition of specific HRE such as Progesterone Response Elements (PRE). In this paper, a comprehensive survey and comparison of in silico methods is conducted for establishing a more accurate statistic model. Homogeneity of steroid HRE is analyzed and a reliable training dataset is constructed through extensive searching for experimentally validated response elements from more than 150 literature sources. Based on the observation that the verified HREs carry di-nucleotide preservation in comparison with uniform nucleotide distributions, both mono and di-nucleotide Position Weight Matrices are computed to extract the statistic pattern of the positions. It is followed by the sequence transition pattern recognition using a specifically designed profile Hidden Markov Model. Reciprocal combination of the statistic and transition patterns significantly improves the performance of the model in terms of higher sensitivity and specificity. Upon acquisition of the putative response elements in the promoter areas of vertebrate genes, a qualitative scheme is applied to assess the probability for each gene to be a hormone primary target. Using >650 records of experimentally validated steroid hormone response elements, a high sensitivity level of 73% and high specificity level of one prediction per 8.24 kb is reached, allowing this model to be used for further prediction of primary target genes through the analysis of their upstream promoters, for human or other vertebrate genomes of interest. Additional documents, supplementary data and the web-based program developed for response elements prediction are freely available for academic research at . Submission of putative gene promoter regions for recognition of potential regulatory PREs can be as long as 5 kb.

FK506-Binding Protein 52 Is Essential to Uterine Reproductive Physiology Controlled by the Progesterone Receptor A Isoform

FK506-binding protein 52 (FKBP52) is a tetratricopeptide repeat protein that associates with steroid receptors in complexes containing heat shock protein 90. To investigate the role of FKBP52 in steroid-regulated physiology, we generated FKBP52-deficient mice. FKBP52 (-/-) females are sterile due to a complete failure of implantation, a process that requires estrogen (ER) and progesterone receptors (PR). Because the uterus expresses two forms of PR, PR-A and PR-B, we investigated all three receptors as potential targets of FKBP52 action. FKBP52 (-/-) uteri showed a normal growth response to estradiol, and unaltered expression of genes controlled by ER and PR-B. In contrast, FKBP52 (-/-) uteri were neither able to express two PR-A-regulated genes, nor undergo decidualization in response to progesterone, suggesting that FKBP52 specifically regulates PR-A at this organ. Analysis of uterine PR heterocomplexes showed preferential association of FKBP52 with PR-A compared with PR-B. Loss of FKBP52 neither disrupted the PR-A/heat shock protein 90 interaction, nor impaired uterine PR-A hormone-binding function, demonstrating the essential role of FKBP52 in PR-A action to be downstream of the hormone-binding event. Transcription studies in +/+ and -/- mouse embryonic fibroblast cells showed a near-complete loss of PR-A activity at mouse mammary tumor virus and synthetic progesterone response element promoters, although partial reductions of ER and PR-B were also observed. Partial disruptions of ovulation and mammary development were also found in FKBP52 (-/-) females. Taken as a whole, our results show FKBP52 to be an essential regulator of PR-A action in the uterus, while being a nonessential but contributory regulator of steroid receptors in the mammary and ovary. These data may now provide the basis for selective targeting of steroid-regulated physiology through tetratricopeptide repeat proteins.