Chicken Ovalbumin Upstream Promoter-transcription Factor I Research Articles

COUP-TFI deletion affects angiogenesis and apoptosis related gene expression in mouse placenta: results of an explorative study

Background: Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI) is a member of the steroid/thyroid nuclear receptor superfamily. The aim of this study was to investigate whether absence of this gene affects placental development and fetal growth in a COUP-TFI knockout mouse model. Methods: Placentas of COUP-TFI-knockout (COUP-TFI KO) and wild-type (WT) were collected at 18.5 days post-coitum. The expression level of the following genes known to be involved in different key molecular pathways was evaluated: BCL2 Associated X (Bax) and B-cell lymphoma 2 (Bcl-2) (apoptosis), p21, p53 and α subunit of inhibin (INHA) (proliferation and apoptosis), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), hypoxia-inducible factor 1-alpha (HIF1α), Fms related receptor tyrosine kinase 1 (Flt-1), and endoglin (ENG) (angiogenesis). Mouse litter weight at birth was also assessed. Results: RT-qPCR analysis showed increased mRNA expression of VEGF-A and Bax in placental tissue of COUP-TFI KO mice compared to WT mice. We also found a loss in the positive correlation between Bcl-2 and INHA, p21 and ENG, as well as HIF1α and Flt-1 mRNA expression in COUP-TFI mutants. Finally, KO mice were lighter than WT littermates (respectively, the mean weight of COUP-TFI KO mice was 1.3 grams, ± 0.13, compared to 1.6 g, ± 0.14 of WT mice, p < 0.05). Conclusions: Our results show that COUP-TFI deletion is associated with a lower birth weight in mice and increased placental transcript expression of pro-apoptotic Bax and pro-angiogenetic VEGF-A genes.

Activation of COUP-TFI by a Novel Diindolylmethane Derivative.

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan receptor and member of the nuclear receptor superfamily. Among a series of methylene substituted diindolylmethanes (C-DIMs) containing substituted phenyl and heteroaromatic groups, we identified 1,1-bis(3′-indolyl)-1-(4-pyridyl)-methane (DIM-C-Pyr-4) as an activator of COUP-TFI. Structure activity studies with structurally diverse heteroaromatic C-DIMs showed that the pyridyl substituted compound was active and the 4-pyridyl substituent was more potent than the 2- or 3-pyridyl analogs in transactivation assays in breast cancer cells. The DIM-C-Pyr-4 activated chimeric GAL4-COUP-TFI constructs containing full length, C- or N-terminal deletions, and transactivation was inhibited by phosphatidylinositol-3-kinase and protein kinase A inhibitors. However, DIM-C-Pyr-4 also induced transactivation and interactions of COUP-TFI and steroid receptor coactivators-1 and -2 in mammalian two-hybrid assays, and ligand-induced interactions of the C-terminal region of COUP-TFI were not affected by kinase inhibitors. We also showed that DIM-C-Pyr-4 activated COUP-TFI-dependent early growth response 1 (Egr-1) expression and this response primarily involved COUP-TFI interactions with Sp3 and to a lesser extent Sp1 bound to the proximal region of the Egr-1 promoter. Modeling studies showed interactions of DIM-C-Pyr-4 within the ligand binding domain of COUP-TFI. This report is the first to identify a COUP-TFI agonist and demonstrate activation of COUP-TFI-dependent Egr-1 expression.

Inhibition of Cell Growth by Nuclear Receptor COUP-TFI: Possible Involvement of Decorin in Growth Inhibition

The transcription factor, Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI) is a member of the steroid/thyroid hormone receptor superfamily and plays important roles especially in development and differentiation. To investigate the effects of COUP-TFI on cell growth, we transfected this factor stably into Ha-rastransformed NIH3T3 (ras-NIH3T3) cells. Ectopic expression of COUP-TFI resulted in reduction of both proliferation rates and anchorage-independent growth. Flow cytometric analysis showed that progression through mid-S to G2/M phase was delayed in COUP-TFI transfectants. cDNA microarray analysis was performed to elucidate candidate genes whose expression levels were changed in response to COUP-TFI introduction. mRNA levels of decorin and cyclin G were elevated in COUP-TFI transfectants. Luciferase reporter assay revealed that the upstream promoter regions between –252 and -205 bp and between -204 and +42 bp of the decorin gene were responsible for COUPTFI. These suggest that decorin transactivated by COUP-TFI is one of the molecules which account for the growthinhibitory effects of COUP-TFI.

Role of COUP‐TFI during retinoic acid‐induced differentiation of P19 cells to endodermal cells

Retinoic acid (RA) is a positive regulator of P19 cell differentiation. Silencing of pre-B cell leukemia transcription factors (PBXs) expression in P19 cells (AS cells) results in a failure of these cells to differentiate to endodermal cells upon RA treatment. Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) is an orphan member of the steroid-thyroid hormone superfamily. RA treatment of wild type P19 cells results in a dramatic increase in the expression of COUP-TFI; however, COUP-TFI mRNA levels fail to be elevated upon RA treatment of AS cells indicating that PBX expression is required for elevation in COUP-TFI expression. To study the role of COUP-TFI during RA-dependent differentiation of P19 cells, AS cells that inducibly express various levels of COUP-TFI were prepared. Exogenous expression of COUP-TFI in AS cells, in a dose-dependent fashion, leads to growth inhibition, modest cell cycle disruption, and early apoptosis. Furthermore, AS cells can overcome the blockage in RA-dependent differentiation to endodermal cells when either pharmacological levels of COUP-TFI are expressed or a combination of both the expression of physiological levels of COUP-TFI and RA treatment. Additionally, the mRNA level of several pluripotency associated genes including OCT-4, DAX-1, and SF-1 in the COUP-TFI expressing AS cells are reduced. Moreover, analysis of the expression of primary RA response genes indicates that COUP-TFI is involved in the regulatory modulation of the expression of at least two genes, CYP26A1 and HoxA1. These studies demonstrate that COUP-TFI functions as a physiologically relevant regulator during RA-mediated endodermal differentiation of P19 cells.

Generation of ES Cells for Conditional Expression of Nuclear Receptors and Coregulatorsin Vivo

Nuclear receptors and coregulators orchestrate diverse aspects of biological functions and inappropriate expression of these factors often associates with human diseases. The present study describes a conditional overexpression system consisting of a minigene located at the Rosa26 locus in the genome of mouse embryonic stem (ES) cells. Before activation, the minigene is silent due to a floxed STOP cassette inserted between the promoter and the transgene. Upon cre-mediated excision of the STOP cassette, the minigene constitutively expresses the tagged transgene driven by the ubiquitous CAGGS promoter. Thus, this system can be used to express target gene in any tissue in a spatial and/or temporal manner if respective cre mouse lines are available. Serving as proof of principle, the CAG-S-hCOUP-TFI allele was generated in ES cells and subsequently in mice. This allele was capable of conditionally overexpressing human chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) in all tissues tested upon activation by cre drivers. This allele was further subjected to address functionality of expressed COUP-TFI and the functional similarity between COUP-TFI and COUP-TFII. Expression of COUP-TFI in COUP-TFII-ablated uterus suppressed aberrant estrogen receptor-alpha activities and rescued implantation and decidualization defects of COUP-TFII mutants, suggesting that COUP-TFI and COUP-TFII are able to functionally compensate for each other in the uterus. A toolbox currently under construction will contain ES cell lines for overexpressing all 48 nuclear receptors and selected 10 coregulators. Upon completion, it will be a very valuable resource for the scientific community. Several ES cells are currently available for distribution.

ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH‐SY5Y cells: the role of COUP‐TFI

Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RARalpha, and RXRalpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.

A Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) Complex Represses Expression of the Gene Encoding Tumor Necrosis Factor α-induced Protein 8 (TNFAIP8)

The orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) plays key roles in development and homeostasis. A tandem affinity purification procedure revealed that COUP-TFI associated with a number of transcriptional regulatory proteins in HeLa S3 cells, including the nuclear receptor corepressor (NCoR), TIF1beta/KAP-1, HDAC1, and the SWI/SNF family member Brahma. The proapoptotic protein DBC1 was also identified in COUP-TFI complexes. In vitro experiments revealed that COUP-TFI interacted directly with NCoR but in a manner different from that of other nuclear receptors. DBC1 stabilized the interaction between COUP-TFI and NCoR by interacting directly with both proteins. The gene encoding the anti-apoptotic protein TNFAIP8 (tumor necrosis factor alpha (TNFalpha)-induced protein 8) was identified as being repressed by COUP-TFI in a manner that required several of the component proteins of the COUP-TFI complex. Finally, our studies highlight a central role for COUP-TFI in the induction of the TNFAIP8 promoter by TNFalpha. Together, these studies identify a novel COUP-TFI complex that functions as a repressor of transcription and may play a role in the TNFalpha signaling pathways.

Expression of Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI) in Bladder Transitional Cell Carcinoma

An analysis of differentially expressed genes (DEGs) between bladder transitional cell carcinoma (TCC) and the surrounding urothelium to help identify what lies behind the mechanism of multifocal tumor development has not yet been performed. We sought to find a new DEG related to the development of bladder TCC. Thirty-nine bladder TCC tissues paired with normal-appearing urothelium tissues obtained from the same patient were used as subjects. Initially, we compared the messenger RNA (mRNA) profiles between normal-appearing urothelium and TCC tissue of 1 patient by using annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) and selective amplification of family members (SAFM) PCR to identify potential DEGs. To validate the results of the ACP data, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on those of all 39 patients. Among the several DEGs discovered in the ACP data, 1 DEG was chosen as the candidate for the RT-PCR, that is present or markedly upregulated in normal-appearing urothelial tissue compared with TCC tissue. Gene sequence searching revealed that this DEG is chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI). Downregulation of COUP-TFI mRNA expression in TCC tissue compared to normal-appearing urothelium tissue of the same patient, irrespective of tumor stage and grade, was confirmed by RT-PCR in 39 patients. Our results suggest that the loss of COUP-TFI may play a role in the transition from normal epithelium to TCC. Further characterization of the COUP-TFI gene is expected to give us informations about bladder TCC tumorigenesis.

Analysis of the expression of estrogen receptor, progesterone receptor and chicken ovalbumin upstream promoter-transcription factor I in ovarian epithelial cancers and normal ovaries

Sex hormones are involved in the carcinogenesis of some gynecologic cancers, and the status of their receptors represents an indicator of prognosis and of the therapeutic response in breast and endometrial cancers. In the ovary, this role is not clearly defined, with epithelial cancers being poorly responsive to hormone therapy. COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) is an orphan nuclear receptor, which is expressed in various tissues and regulates the estrogen receptor (ER) by competition for DNA binding. To investigate the role of these receptors in ovarian carcinogenesis and their implications for cancer prognosis, we evaluated the immunohistochemical expression of ER, progesterone receptor (PR) and COUP-TFI in benign and malignant ovarian epithelial neoplasms and in normal ovaries. A total of 113 ovarian specimens, including 40 diagnosed as malignant epithelial neoplasms (group A), 45 as benign epithelial tumors (group B), and 28 from normal ovaries (group C) were analyzed. Immunoexpression of ER was observed in 70% of patients of group A, 57.8% of group B and 57.1% of group C, with no significant difference between groups (p=0.426). Immunoexpression of PR was significantly lower in group A (12.5%) compared to group B (42.2%) and group C (32.1%) (p=0.010). Similarly, COUP-TFI was expressed in only 10% of group A patients, a rate significantly lower than that observed for group B (31.1%) and group C (39.3%) (p=0.014). No association was observed between the expression of these markers and increased survival or clinical prognostic variables. Multivariate analysis revealed a residual tumor <1 cm as the most significant clinical prognostic factor in group A (p=0.010, OR=4.14). These data support the importance of cytoreduction in the treatment of ovarian cancer, the role of steroid receptors in the mechanism of carcinogenesis, and the need for selection of subgroups that may respond to hormone therapy.

Deoxyribonucleic Acid Methyl Transferases 3a and 3b Associate with the Nuclear Orphan Receptor COUP-TFI during Gene Activation

Transcriptional activation of silent genes can require the erasure of epigenetic marks such as DNA methylation at CpGs (cytosine-guanine dinucleotide). Active demethylation events have been observed, and associated processes are repeatedly suspected to involve DNA glycosylases such as mCpG binding domain protein 4, thymine DNA glycosylase (TDG), Demeter, and repressor of silencing 1. A complete characterization of the molecular mechanisms occurring in metazoan is nonetheless awaited. Here, we report that activation of the endogenous vitronectin gene in P19 cells by the nuclear receptor chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is observed in parallel with the recruitment of TDG and p68 RNA helicase, two components of a putative demethylation complex. Interestingly, when activated, the vitronectin gene was loaded with DNA methyltransferases 3a and 3b (Dnmt3a/b), and a strand-biased decrease in CpG methylation was detected. Dnmt3a was further found to associate with COUP-TFI and TDG in vivo, and cotransfection experiments demonstrated that Dnmt3a/b can enhance COUP-TFI-mediated activation of a methylated reporter gene. These results suggest that Dnmt3a/b could cooperate with the orphan receptor COUP-TFI to regulate transcription of the vitronectin gene.

Increased Retinoic Acid Receptor-β4 Correlates In vivo with Reduced Retinoic Acid Receptor-β2 in Esophageal Squamous Cell Carcinoma

Different retinoic acid receptor-beta (RAR-beta) isoforms seem to have contrasting biological effects in human carcinogenesis. Both in vitro and in vivo data indicate that RAR-beta2 expression is frequently lost or reduced (and transfecting RAR-beta2 suppresses growth and promotes apoptosis) in various cancer cells and tissues, whereas RAR-beta4 expression is increased in several cancer cell lines. To clarify the effects of different RAR-beta isoforms in esophageal carcinogenesis, we used real-time quantitative reverse transcription-PCR to assess in vivo RAR-beta mRNA levels in specimens of normal and malignant human esophageal tissue, comparing these levels with each other and the expressions of other genes. RAR-beta2 mRNA expression was significantly reduced (i.e., lower in cancer than normal tissue) in 67% (18 of 27, P = 0.001) and RAR-beta(4) mRNA was increased in 52% (14 of 27, P = 0.054) of our esophageal cancer cases. The expressions of RAR-beta1, chicken ovalbumin upstream promoter-transcription factor-I (COUP-TFI), COUP-TFII, and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) mRNA were reduced, whereas epidermal growth factor receptor and cyclin D1 expressions were increased in tumor compared with in normal tissues. Reduced RAR-beta2 expression correlated with increased RAR-beta4 expression (P = 0.002) and with the suppression of COUP-TFI and COUP-TFII (P = 0.050 and 0.023, respectively) in tumor samples. These are the first in vivo expression patterns of RAR-beta2 and RAR-beta4 reported in humans or animals and support the in vitro data on these isoforms and their contrasting biological effects in human carcinogenesis.

Effects of the COUP-TFI mutation on murine cochlear function

Problem: COUP-TFI (Chicken Ovalbumin Upstream Promoter-Transcription Factor I) is an orphan member of the steroid/thyroid nuclear receptor superfamily. It is highly expressed in the developing nervous system. Null mutant COUP-TFI mice have glossopharyngeal nerve dysfunction that compromises feeding ability and usually results in perinatal death. Surviving null mutant mice demonstrate neuronal differentiation defects, altered thalamocortical axon guidance, and apoptosis of cerebral cortical layer IV. COUP-TFI expression is also important for inner ear organogenesis. Null mutants have a shortened cochlear duct, abnormal cochlear innervation, and malformed vestibular chambers. Additionally, by P20 the organ of Corti in the basal turn has degenerated. Interestingly, the organ of Corti at the mid-modiolar region has only subtle anatomic irregularities and, in particular, contains normal-appearing inner and outer hair cells. We sought to determine the effect of this mutation on cochlear function. Methods: We recorded ABR and DPOAE thresholds in P16-18 null mutant and heterozygote COUP TFI mice, as well as age-matched wild-type controls. ABRs were evoked with a 5 msec sine wave tone pip. DPOAEs were elicited using F2 = 1.2*F1 and L2 = L1. All measurements were performed using stimuli up to 80 dB SPL over the frequency spectrum of 1 to 40 kHz. Results: Null mutant mice had no ABR and no DPOAE responses over the range of stimulation. In contrast, heterozygote and wild-type mice had normal ABR and DPOAE thresholds of 30 to 50 dB in the mid-to-high frequencies. In general, these responses occurred above 6.3 kHz. Conclusion: Both measures of hearing indicate that the COUP-TFI null mutant has profound cochlear dysfunction. Cochlear function in heterozygotes is no different than in wild-type mice. Significance: Even though these mutants have cochleae with many normal anatomic features, these features do not appear to have physiologic relevance. These mutants may be a good model of congenital hearing loss associated with otic capsule malformation. Support: None reported.

Multiple phosphorylation events control chicken ovalbumin upstream promoter transcription factor I orphan nuclear receptor activity.

Chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) is an orphan member of the nuclear hormone receptor superfamily that comprises key regulators of many biological functions, such as embryonic development, metabolism, homeostasis, and reproduction. Although COUP-TFI can both actively silence gene transcription and antagonize the functions of various other nuclear receptors, the COUP-TFI orphan receptor also acts as a transcriptional activator in certain contexts. Moreover, COUP-TFI has recently been shown to serve as an accessory factor for some ligand-bound nuclear receptors, suggesting that it may modulate, both negatively and positively, a wide range of hormonal responses. In the absence of any identified cognate ligand, the mechanisms involved in the regulation of COUP-TFI activity remain unclear. The elucidation of several putative phosphorylation sites for MAPKs, PKC, and casein kinase II within the sequence of this orphan receptor led us to investigate phosphorylation events regulating the various COUP-TFI functions. After showing that COUP-TFI is phosphorylated in vivo, we provide evidence that in vivo inhibition of either MAPK or PKC signaling pathway leads to a specific and pronounced decrease in COUP-TFI-dependent transcriptional activation of the vitronectin gene promoter. Focusing on the molecular mechanisms underlying the MAPK- and PKC-mediated regulation of COUP-TFI activity, we show that COUP-TFI can be directly targeted by PKC and MAPK. These phosphorylation events differentially modulate COUP-TFI functions: PKC-mediated phosphorylation enhances COUP-TFI affinity for DNA and MAPK-mediated phosphorylation positively regulates the transactivation function of COUP-TFI, possibly through enhancing specific coactivator recruitment. These data provide evidence that COUP-TFI is likely to integrate distinct signaling pathways and raise the possibility that multiple extracellular signals influence biological processes controlled by COUP-TFI.

Synergistic activation of the Atlantic salmon hepatocyte nuclear factor (HNF) 1 promoter by the orphan nuclear receptors HNF4 and chicken ovalbumin upstream promoter transcription factor I (COUP-TFI)

Synergistic activation of the Atlantic salmon hepatocyte nuclear factor (HNF) 1 promoter by the orphan nuclear receptors HNF4 and chicken ovalbumin upstream promoter transcription factor I (COUP-TFI)

Synergistic activation of the Atlantic salmon hepatocyte nuclear factor (HNF) 1 promoter by the orphan nuclear receptors HNF4 and chicken ovalbumin upstream promoter transcription factor I (COUP-TFI)

Hepatocyte nuclear factor 1 (HNF1) is a liver-enriched transcription factor that plays an important role in transcriptional networks involved in liver function. The promoters of mammalian HNF1 genes contains a single binding site for another liver-enriched transcription factor, the nuclear hormone receptor HNF4. A transcriptional hierarchy involving HNF4-mediated activation of the HNF1 promoter has been proposed to be of crucial importance in maintaining the differentiated hepatocyte phenotype. Here we present evidence that the Atlantic salmon HNF1 promoter contains three nuclear-hormone-receptor-binding sequences. Gel-shift assays showed that these motifs are recognized with different affinities by HNF4 and the orphan nuclear receptors chicken ovalbumin upstream promoter transcription factors COUP-TFI and COUP-TFII. In hepatoma cells, the site showing highest affinity for HNF4 appears to be crucial for promoter activity. Transfection experiments in non-hepatic cells indicated that the salmon HNF1 promoter was activated by both HNF4 and COUP-TFs. We also identified a promoter fragment encompassing the two more distal nuclear-hormone-binding sites that was activated by HNF4, unaffected by COUP-TF and showed a strong synergistic activation by HNF4/COUP-TF. Results are presented detailing these interactions in relation to the salmon HNF1 promoter architecture.

Inhibition of retinoic acid-inducible transcription by COUP-TFI in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma cells (HSG) express nuclear receptors, all-trans-retinoic acid (at-RA) receptors (RARs), and retinoid X/9-cis-retinoic acid (9-c-RA) receptors (RXRs). In order to investigate whether the endogenous RARs or RXRs of HSG cells can induce transcription activation, the thymidine kinase promoter (TK)-driven luciferase reporter gene containing the retinoic acid response element (RARE), of RARbeta, betaRARE2-TK-Luc, was transfected into HSG cells and ligand-dependent transcription activation was examined. Luciferase activity of cell lysate increased by the treatment with either at-RA or 9-c-RA. Co-transfection of RARalpha and (or) RXRalpha-expression plasmids with the reporter gene enhanced the luciferase activity, suggesting that endogenous RARs and RXRs work as ligand-dependent transfactors in HSG cells. Reverse transcriptase - polymerase chain reaction analysis revealed that HSG cells express chicken ovalbumin upstream promoter - transcription factor I (COUP-TFI). Co-transfection of COUP-TFI-expression plasmid suppressed the at-RA-induced transcription activation of the reporter gene. Similar results were shown using a chromatin-integrated reporter gene system, using a stably transfected beta-RARE2-TK-beta-galactosidase (beta-Gal) reporter gene. The at-RA-dependent increase in the beta-Gal expression was completely inhibited by COUP-TFI. The transfection of antisense oligonucleotide of COUP-TFI squelched the RA-dependent growth inhibition induced by RAR-RXR heterodimers. Conclusively, RARs and RXRs of HSG cells are functional and play roles as transactivators in at-RA-sensitive processes such as the proliferation or differentiation of cells. COUP-TFI very likely regulates these processes by repressing the functions of these transactivators.

Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene.

In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.

COUP-TFI Expression in Human Adrenocortical Adenomas: Possible Role in Steroidogenesis

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan nuclear receptor essential for neurogenesis, organogenesis, and cell fate determination. CYP17 gene transcription has recently been shown to be activated by SF-1 (steroidogenic factor-1) binding to a cyclic AMP-responsive sequence within the promoter region of the gene, and inhibited by COUP-TF binding to the sequence. Thus, COUP-TF and SF-1 act as a transcriptional repressor and activator, respectively, of CYP17 gene expression. Transcriptional repression by COUP-TFI is mediated by corepressors, N-CoR (nuclear receptor-corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone-receptor), whereas transcriptional activation by SF-1 is mediated by coactivator SRC-1 (steriod receptor coactivator-1). We therefore examined the expression of COUP-TFI, SF-1, SRC-1, N-CoR, and SMRT in a variety of adrenocortical adenomas and compared the results with CYP17 mRNA levels. We found significantly high COUP-TFI mRNA expression in nonfunctional adenomas (n=8: 220±16%; normal 96±4%), a deoxycorticosterone-producing adenoma (n=1: 200%), and a pre-clinical Cushing’s adenoma (n=1: 280%), intermediate COUP-TFI expression in cortisol-producing adenomas (n=8: 63±5%), and low COUP-TFI expression in aldosterone-producing adenomas (n=8: 49±4%). In contrast to COUP-TFI, SF-1 mRNA expression did not vary significantly among adrenals. We did not detect the expected negative correlation between COUP-TFI and CYP17 mRNA levels in adrenocortical adenomas. High COUP-TFI expression was associated with a nonfunctioning phenotype. Interestingly, the pattern of COUP-TFI expression was similar to the profile of N-CoR expression, but not of SMRT expression. These results indicate that COUP-TFI and N-CoR may play a role in steroidogenesis by human adrenocortical adenomas.

COUP-TFI expression in human adrenocortical adenomas: possible role in steroidogenesis.

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan nuclear receptor essential for neurogenesis, organogenesis, and cell fate determination. CYP17 gene transcription has recently been shown to be activated by SF-1 (steroidogenic factor-1) binding to a cyclic AMP-responsive sequence within the promoter region of the gene, and inhibited by COUP-TF binding to the sequence. Thus, COUP-TF and SF-1 act as a transcriptional repressor and activator, respectively, of CYP17 gene expression. Transcriptional repression by COUP-TFI is mediated by corepressors, N-CoR (nuclear receptor-corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone-receptor), whereas transcriptional activation by SF-1 is mediated by coactivator SRC-1 (steroid receptor coactivator-1). We therefore examined the expression of COUP-TFI, SF-1, SRC-1, N-CoR, and SMRT in a variety of adrenocortical adenomas and compared the results with CYP17 mRNA levels. We found significantly high COUP-TFI mRNA expression in nonfunctional adenomas (n=8: 220+/-16%; normal 96+/-4%), a deoxycorticosterone-producing adenoma (n=1: 200%), and a pre-clinical Cushing's adenoma (n=1: 280%), intermediate COUP-TFI expression in cortisol-producing adenomas (n=8: 63+/-5%), and low COUP-TFI expression in aldosterone-producing adenomas (n=8: 49+/-4%). In contrast to COUP-TFI, SF-1 mRNA expression did not vary significantly among adrenals. We did not detect the expected negative correlation between COUP-TFI and CYP17 mRNA levels in adrenocortical adenomas. High COUP-TFI expression was associatedwith a nonfunctioning phenotype. Interestingly, the pattern of COUP-TFI expression was similar to the profile of N-CoR expression, but not of SMRT expression. These results indicate that COUP-TFI and N-CoR may play a role in steroidogenesis by human adrenocortical adenomas.

Multiple parameters determine the specificity of transcriptional response by nuclear receptors HNF-4, ARP-1, PPAR, RAR and RXR through common response elements.

A number of nuclear receptors, including retinoic acid receptors (RARs), retinoid-X receptors (RXRs), hepatocyte nuclear factor 4 (HNF-4), chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), apolipoprotein regulatory protein 1 (ARP-1) and peroxisome proliferator-activated receptor (PPAR), bind to response elements comprised of two core motifs, 5'-RG(G/T)TCA, or a closely related sequence separated by 1 nt (DR1 elements). The potential role of the precise sequence of the core motif as well as the spacer nucleotide in determining specificity and promiscuity of receptor-response element interactions was investigated. We show here that nucleotides at base positions 1, 2 and 4 of the core motif as well as the spacer nucleotide determine the binding preference of HNF-4 and ARP-1 homodimers and RAR:RXR and PPAR:RXR heterodimers. In transfection experiments transcriptional activation by HNF-4 and PPAR:RXR and repression by ARP-1 correlated with the relative in vitro binding affinity provided the element was located within the proper promoter context. Furthermore, promoter context also determined whether an element that binds to HNF-4 and PPAR:RXR with equal affinity functions as an HNF-4 response element or PPAR response element. Thus, apart from the element-specific differences in affinity for the receptors, additional promoter-specific transcription factors that interact with HNF-4 and PPAR:RXR determine the specificity of transcriptional response through DR1-type elements.