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1.  Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression. 
Molecular and Cellular Biology  1997;17(9):4914-4932.
The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed deletion analysis of the protein was performed. Chimeric proteins in which various segments of the ARP-1/COUP-TFII carboxy terminus were fused to the GAL4 DNA binding domain were used to characterize its active repression domain. The smallest segment confering active repressor activity to a heterologous DNA binding domain was found to comprise residues 210 to 414. This domain encompasses the region of ARP-1/COUP-TFII corresponding to helices 3 to 12 in the recently published crystal structure of other members of the nuclear receptor superfamily. It includes the AF-2 AD core domain formed by helix 12 but not the hinge region, which is essential for interaction with a corepressor in the case of the thyroid hormone and retinoic acid receptor. Attachment of the nuclear localization signal from the simian virus 40 large T antigen (Flu tag) to the amino terminus of ARP-1/COUP-TFII abolished its ability to bind to DNA without affecting its repressor activity. By using a series of Flu-tagged mutants, the domains required for transrepressor activity of the protein were mapped. They include the DNA binding domain and the segment spanning residues 193 to 399. Transcriptional activity induced by liver-enriched transactivators such as hepatocyte nuclear factor 3 (HNF-3), C/EBP, or HNF-4 was repressed by ARP-1/COUP-TFII independent of the presence of its cognate binding site, while basal transcription or transcriptional activity induced by ATF or Sp1 was not perturbed by the protein. In conclusion, our results demonstrate that the domains of ARP-1/COUP-TFII required for active repression and transrepression do not coincide. Moreover, they strongly suggest that transrepression is the predominant mechanism underlying repressor activity of ARP-1/COUP-TFII. This mechanism most likely involves interaction of the protein with one or several transcriptional coactivator proteins which are employed by various liver-enriched transactivators but not by ubiquitous factors such as Sp1 or ATF.
PMCID: PMC232344  PMID: 9271371
2.  A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in embryonal carcinoma cells. 
Molecular and Cellular Biology  1995;15(2):1034-1048.
The Oct-3/4 transcription factor is a member of the POU family of transcription factors and, as such, probably plays a crucial role in mammalian embryogenesis and differentiation. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Similarly, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repressed in retinoic acid (RA)-differentiated EC cells. Previously we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for negative regulators. Our present results demonstrate that in P19 and RA-treated P19 cells, the orphan receptors ARP-1/COUP-TFII and EAR-3/COUP-TFI repress Oct-3/4 promoter activity through the RAREoct site in a dose-dependent manner. While the N-terminal region of the ARP-1/COUP-TFII receptor is dispensable for this repression, the C-terminal domain harbors the silencing region. Interestingly, three different RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha:RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bind and activate Oct-3/4 promoter through the RAREoct site in a ligand-dependent manner. We have shown that antagonism between ARP-1/COUP-TFII or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellular balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repression by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RAR:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by ARP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affinity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to compete with and even displace RAR:RXR from the RAREoct site and subsequently to actively silence the Oct-3/4 promoter. We have shown that RA treatment of EC cells results in up-regulation of ARP-1/COUP-TFII and EAR-3/COUP-TFI expression. Most interestingly, in RA-treated EC cells, the kinetics of Oct-3/4 repression inversely correlates with the kinetics of ARP-1/COUP-TFII and EAR-3/COUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors participate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene expression during development.
PMCID: PMC232002  PMID: 7823919
3.  The MODY1 Gene for Hepatocyte Nuclear Factor 4α and a Feedback Loop Control COUP-TFII Expression in Pancreatic Beta Cells▿  
Molecular and Cellular Biology  2008;28(14):4588-4597.
Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with the heterozygous deletion of the gene for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) or the complete disruption of the gene for hepatocyte nuclear factor 4α (HNF4α) in pancreatic beta cells have similar insulin secretion defects, leading us to hypothesize that there is transcriptional cross talk between these two nuclear receptors. Here, we demonstrate specific HNF4α activation of a reporter plasmid containing the COUP-TFII gene promoter region in transfected pancreatic beta cells. The stable association of the endogenous HNF4α with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4α transactivation of COUP-TFII. The dominant negative suppression of HNF4α function decreased endogenous COUP-TFII expression, and the specific inactivation of COUP-TFII by small interfering RNA caused HNF4α mRNA levels in 832/13 INS-1 cells to decrease. This positive regulation of HNF4α by COUP-TFII was confirmed by the adenovirus-mediated overexpression of human COUP-TFII (hCOUP-TFII), which increased HNF4α mRNA levels in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression, as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore, COUP-TFII may contribute to the control of insulin secretion through the complex HNF4α/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.
doi:10.1128/MCB.01191-07
PMCID: PMC2447131  PMID: 18474611
4.  Identification and Characterization of Nucleolin as a COUP-TFII Coactivator of Retinoic Acid Receptor β Transcription in Breast Cancer Cells 
PLoS ONE  2012;7(5):e38278.
Introduction
The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.
Results
FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.
Conclusions
Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.
doi:10.1371/journal.pone.0038278
PMCID: PMC3365040  PMID: 22693611
5.  Chicken ovalbumin upstream promoter transcription factor II regulates uncoupling protein 3 gene transcription in Phodopus sungorus 
Background
Ucp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents.
Results
By quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816–840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays.
Conclusion
Transcriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism.
doi:10.1186/1471-2199-8-1
PMCID: PMC1779797  PMID: 17204145
6.  Positive regulation of the vHNF1 promoter by the orphan receptors COUP-TF1/Ear3 and COUP-TFII/Arp1. 
Molecular and Cellular Biology  1996;16(3):778-791.
vHNF1 (also termed HNF1 beta) is a member of the hepatocyte nuclear fa ctor 1 (HNF1; also termed HNF1 alpha) family of homeodomain-containing transcription factors that interact with a sequence motif found in the regulatory regions of a large number of genes expressed mainly in the liver. It has been suggested that vHNF1 plays a role in early differentiation of specialized epithelia of several endoderm- and mesoderm-derived organs, with HNF1 playing a role in later stages. In support of this idea, expression of vHNF1 but not HNF1 is induced upon treatment of the embryonal carcinoma cell line F9 with retinoic acid. We have cloned and analyzed the vHNF1 promoter to gain a better understanding of the regulation of vHNF1 expression and how it relates to the expression of HNF1. We have identified five sites of DNA-protein interaction within the first 260 bp upstream of the transcription start site, which involve at least three different families of transcription factors. Two sites, a distal DR-1 motif and a proximal octamer motif, are the most important for promoter activity. The DR-1 motif interacts with several members of the steroid hormone receptor superfamily including HNF4, COUP-TFI/Ear3, COUP-TFII/Arp1, and RAR alpha/RXR alpha heterodimers. The vHNF1 promoter is transactivated by COUP-TFI/Ear3 and COUP-TFII/Arp1 and, unlike the HNF1 promoter, is virtually unaffected by HNF4. Interestingly, the proximal octamer site and not the DR-1 site is required for COUP-TFI/Ear3 and COUP-TFII/Arp1 transactivation of the vHNF1 promoter. COUP-TFI/Ear3 does not bind directly to this proximal octamer site. We present evidence of an interaction between COUP-TFI/Ear3 and the octamer-binding proteins in vitro and in the cell, suggesting that COUP-TFI and COUP-TFII activate the vHNF1 promoter via an indirect mechanism.
PMCID: PMC231058  PMID: 8622679
7.  COUP-TFII Mediates Progesterone Regulation of Uterine Implantation by Controlling ER Activity 
PLoS Genetics  2007;3(6):e102.
Progesterone and estrogen are critical regulators of uterine receptivity. To facilitate uterine remodeling for embryo attachment, estrogen activity in the uterine epithelia is attenuated by progesterone; however, the molecular mechanism by which this occurs is poorly defined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2), a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma and its expression is regulated by the progesterone–Indian hedgehog–Patched signaling axis that emanates from the epithelium. To further assess COUP-TFII uterine function, a conditional COUP-TFII knockout mouse was generated. This mutant mouse is infertile due to implantation failure, in which both embryo attachment and uterine decidualization are impaired. Using this animal model, we have identified a novel genetic pathway in which BMP2 lies downstream of COUP-TFII. Epithelial progesterone-induced Indian hedgehog regulates stromal COUP-TFII, which in turn controls BMP2 to allow decidualization to manifest in vivo. Interestingly, enhanced epithelial estrogen activity, which impedes maturation of the receptive uterus, was clearly observed in the absence of stromal-derived COUP-TFII. This finding is consistent with the notion that progesterone exerts its control of implantation through uterine epithelial-stromal cross-talk and reveals that stromal-derived COUP-TFII is an essential mediator of this complex cross-communication pathway. This finding also provides a new signaling paradigm for steroid hormone regulation in female reproductive biology, with attendant implications for furthering our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in such human reproductive disorders as endometriosis and endometrial cancer.
Author Summary
Pregnancy is established and maintained through a series of precisely choreographed cellular and molecular events that are controlled by two sex hormones, estrogen and progesterone. Both hormones exert their actions through their distinct nuclear receptors. During the peri-implantation period, estrogen activity is attenuated by progesterone to facilitate epithelial remodeling and embryo attachment, but the detailed molecular mechanism of how this process is achieved remains largely undefined. COUP-TFII (chicken ovalbumin upstream promoter transcription factor II; also known as NR2F2), a member of the nuclear receptor superfamily, is highly expressed in the uterine stroma, and its expression is controlled by progesterone–Indian hedgehog–Patched signaling from the epithelium to the stroma. To assess the uterine function of COUP-TFII, uterine-specific COUP-TFII knockout mice were generated. These mutant mice are infertile due to failure of implantation. We identified a novel genetic pathway in which the epithelial Ihh regulates the stroma COUP-TFII to control BMP2 and regulates decidualization. Interestingly, enhanced epithelial estrogen activity, which impedes the maturation of receptive uterus, was clearly noted in the absence of COUP-TFII. This finding reveals that COUP-TFII plays a critical role in maintaining the balance between estrogen and progesterone activities to establish proper implantation. This finding also provides new insights into women's health care associated with uncontrolled estrogen activity, such as breast cancer and endometriosis.
doi:10.1371/journal.pgen.0030102
PMCID: PMC1892047  PMID: 17590085
8.  COUP-TF Upregulates NGFI-A Gene Expression through an Sp1 Binding Site 
Molecular and Cellular Biology  1999;19(4):2734-2745.
The formation of various tissues requires close communication between two groups of cells, epithelial and mesenchymal cells. COUP-TFs are transcription factors which have been shown to have functions in embryonic development. COUP-TFI is expressed mainly in the nervous system, and its targeted deletion leads to defects in the central and peripheral nervous systems. COUP-TFII is highly expressed in the mesenchymal component of the developing organs. A null mutation of COUP-TFII results in the malformation of the heart and blood vessels. From their expression pattern, we proposed that COUP-TFs regulate paracrine signals important for mesenchymal cell-epithelial cell interactions. In order to identify genes regulated by COUP-TF in this process, a rat urogenital mesenchymal cell line was stably transfected with a COUP-TFI expression vector. We found that NGFI-A, a gene with important functions in brain, organ, and vasculature development, has elevated mRNA and protein levels upon overexpression of COUP-TFI in these cells. A study of the promoter region of this gene identified a COUP-TF-responsive element between positions −64 and −46. Surprisingly, this region includes binding sites for members of the Sp1 family of transcription factors but no COUP-TF binding site. Mutations that abolish the Sp1 binding activity also impair the transactivation of the NGFI-A promoter by COUP-TF. Two regions of the COUP-TF molecule are shown to be important for NGFI-A activation: the DNA binding domain and the extreme C terminus of the putative ligand binding domain. The C-terminal region is likely to be important for interaction with coactivators. In fact, the coactivators p300 and steroid receptor activator 1 can enhance the transactivation of the NGFI-A promoter induced by COUP-TFI. Finally, we demonstrated that COUP-TF can directly interact with Sp1. Taken together, these results suggest that NGFI-A is a target gene for COUP-TFs and that the Sp1 family of transcription factors mediates its regulation by COUP-TFs.
PMCID: PMC84066  PMID: 10082539
9.  The orphan nuclear receptor, COUP-TF II, inhibits myogenesis by post-transcriptional regulation of MyoD function: COUP-TF II directly interacts with p300 and myoD. 
Nucleic Acids Research  1998;26(23):5501-5510.
COUP-TF II is an orphan nuclear receptor that has no known ligand in the 'classical sense'. COUP-TF interacts with the corepressors N-CoR, SMRT and RIP13, and silences transcription by active repression and trans-repression. Forced expression of the orphan nuclear receptor COUP-TF II in mouse C2 myogenic cells has been demonstrated to inhibit morphological differentiation, and to repress the expression of: (i) the myoD gene family which encodes myogenic basic helix-loop-helix (bHLH) proteins; and (ii) the cell cycle regulator, p21(Waf-1/Cip-1). In the present study, we show that COUP-TF II efficiently inhibits the myoD -mediated myogenic conversion of pluripotential C3H10T1/2 cells by post-transcriptional mechanisms. Furthermore, repression of MyoD-dependent transcription by COUP-TF II occurs in the absence of the nuclear receptor cognate binding motif. The inhibition of MyoD-mediated trans-activation involves the direct binding of the DNA binding domain/C-region and hinge/D-regions [i.e. amino acid (aa) residues 78-213] of COUP-TF II to the N-terminal activation domain of MyoD. Over-expression of the cofactor p300, which functions as a coactivator of myoD-mediated transcription, alleviated repression by COUP-TF II. Further binding analysis demonstrated that COUP-TF II interacted with the N-terminal 149 aa residues of p300 which encoded the receptor interaction domain of the coactivator. Finally we observed that COUP-TF II, MyoD and p300 interact in a competitive manner, and that increasing amounts of COUP-TF II have the ability to reduce the interaction between myoD and p300 invitro. The experiments presented herein suggest thatCOUP-TF II post-transcriptionally regulates myoD activity/function, and that crosstalk between orphan nuclear receptors and the myogenic bHLH proteins has functional consequences for differentiation.
PMCID: PMC147985  PMID: 9826778
10.  Chicken ovalbumin upstream promoter transcription factors act as auxiliary cofactors for hepatocyte nuclear factor 4 and enhance hepatic gene expression. 
Molecular and Cellular Biology  1997;17(5):2790-2797.
Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) strongly inhibit transcriptional activation mediated by nuclear hormone receptors, including hepatocyte nuclear factor 4 (HNF-4). COUP-TFs repress HNF-4-dependent gene expression by competition with HNF-4 for common binding sites found in several regulatory regions. Here we show that promoters, such as the HNF-1 promoter, which are recognized by HNF-4 but not by COUP-TFs are activated by COUP-TFI and COUP-TFII in conjunction with HNF-4 more than 100-fold above basal levels, as opposed to about 8-fold activation by HNF-4 alone. This enhancement was strictly dependent on an intact HNF-4 E domain. In vitro and in vivo evidence suggests that COUP-TFs enhance HNF-4 activity by a mechanism that involves their physical interaction with the amino acid 227 to 271 region of HNF-4. Our results indicate that in certain promoters, COUP-TFs act as auxiliary cofactors for HNF-4, orienting the HNF-4 activation domain in a more efficient configuration to achieve enhanced transcriptional activity. These findings provide new insights into the regulatory functions of COUP-TFs, suggesting their involvement in the initial activation and subsequent high-level expression of hepatic regulators, as well as in the positive and negative modulation of downstream target genes.
PMCID: PMC232130  PMID: 9111350
11.  Essential Roles of COUP-TFII in Leydig Cell Differentiation and Male Fertility 
PLoS ONE  2008;3(9):e3285.
Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII; also known as NR2F2), is an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily. COUP-TFII-null mice die during the early embryonic development due to angiogenesis and cardiovascular defects. To circumvent the early embryonic lethality and investigate the physiological function of COUP-TFII, we knocked out COUP-TFII gene in a time-specific manner by using a tamoxifen inducible Cre recombinase. The ablation of COUP-TFII during pre-pubertal stages of male development results in infertility, hypogonadism and spermatogenetic arrest. Homozygous adult male mutants are defective in testosterone synthesis, and administration of testosterone could largely rescue the mutant defects. Notably, the rescued results also provide the evidence that the major function of adult Leydig cell is to synthesize testosterone. Further phenotypic analysis reveals that Leydig cell differentiation is arrested at the progenitor cell stage in the testes of null mice. The failure of testosterone to resumption of Leydig cell maturation in the null mice indicates that COUP-TFII itself is essential for this process. In addition, we identify that COUP-TFII plays roles in progenitor Leydig cell formation and early testis organogenesis, as demonstrated by the ablation of COUP-TFII at E18.5. On the other hand, when COUP-TFII is deleted in the adult stage after Leydig cells are well differentiated, there are no obvious defects in reproduction and Leydig cell function. Taken together, these results indicate that COUP-TFII plays a major role in differentiation, but not the maintenance of Leydig cells.
doi:10.1371/journal.pone.0003285
PMCID: PMC2553269  PMID: 18818749
12.  The nuclear orphan receptors COUP-TF and ARP-1 positively regulate the trout estrogen receptor gene through enhancing autoregulation. 
Molecular and Cellular Biology  1997;17(9):5053-5066.
The rainbow trout estrogen receptor (rtER) is a positively autoregulated gene in liver cells. In a previous report, we showed that upregulation is mediated by an estrogen response element (ERE) located in the proximal promoter of the gene and that a half binding site for nuclear receptors (5'-TGACCT-3') located 15 bp upstream of the ERE is involved in the magnitude of the estrogen response. We now report that the human orphan receptor COUP-TF and a COUP-TF-like protein from trout liver are able to bind to the consensus half-site. When cotransfected with the rtER gene proximal promoter, COUP-TF had no regulatory functions on its own. Interestingly, COUP-TF enhanced rtER transactivation properties in the presence of estradiol in a dose-dependent manner when cotransfected with the rtER gene promoter. Unliganded retinoid receptor heterodimers had the same helper function as COUP-TF in the presence of estradiol but were switched to repressors when the ligand all-trans-retinoic acid was added. Mutation of the consensus half-site only slightly reduced COUP-TF helper function, suggesting that it actually results from a complex mechanism that probably involves both DNA binding of COUP-TF to the promoter and protein-protein interaction with another transcription factor bound to the promoter. Nevertheless, a DNA-binding-defective mutant of COUP-TF was also defective in ER helper function. Competition footprinting analysis suggested that COUP-TF actually establishes contacts with the consensus upstream half-site and the downstream ERE half-site that would form a DR-24-like response element. Interaction of COUP-TF with the DR-24 element was confirmed in footprinting assays by using nuclear extracts from Saccharomyces cerevisiae expressing COUP-TF. Finally, interaction of COUP-TF with mutants of the rtER gene promoter showed that COUP-TF recognizes the ERE when the upstream half-site is mutated. These data show that COUP-TF may activate transcription through interaction with other nuclear receptors. This cross-talk between liganded nuclear receptors and orphan receptors is likely to modulate the spectrum of action of a particular ligand-receptor complex and may participate in the cell-type specificity of the ligand effect.
PMCID: PMC232356  PMID: 9271383
13.  The Nutritional Induction of COUP-TFII Gene Expression in Ventromedial Hypothalamic Neurons Is Mediated by the Melanocortin Pathway 
PLoS ONE  2010;5(10):e13464.
Background
The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues.
Methodology/Principal Findings
Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression.
Conclusions/Significance
These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation.
doi:10.1371/journal.pone.0013464
PMCID: PMC2956692  PMID: 20976162
14.  Haploinsufficiency of COUP-TFII in Female Reproduction 
The chicken ovalbumin upstream promoter transcription factor II, COUP-TFII, is a member of the Orphan nuclear receptor transcription factor family. Genetic ablation of COUP-TFII results in early embryonic lethality and demonstrates that this gene is required for cardiac and vascular development. Expression of COUP-TFII persists throughout postnatal life in various tissues including the female reproductive tract. However, the physiological function of COUP-TFII in female reproduction has not been extensively analyzed. Here, we provide phenotypic evidences that haploinsufficiency of COUP-TFII in mice demonstrates an important role of COUP-TFII for normal female reproduction. COUP-TFII +/− females show significantly reduced fecundity, irregular estrus cycles, delayed puberty and retarded postnatal growth. Analysis of the reduced fertility revealed that although ovarian function was normal with respect to ovulation, the ovaries have reduced ability to synthesize progesterone in response to exogenous gonadotropins. This reduction is due to the reduction of the expression of steroidogenic enzymes important for P4 synthesis and the reduction of vascularization in COUP-TFII heterozygotes. Analysis of uterine function demonstrated a reduced response to an experimentally induced decidual cell reaction indicating that the ability of the uterus to support embryo implantation was reduced. Taken together, our data shows global impact of gene dosage effects of COUP-TFII on female postnatal life and indicates requirement of COUP-TFII in normal female reproduction, in particular for uterine endometrial functions during the peri-implantation period.
doi:10.1210/me.2005-0019
PMCID: PMC1198323  PMID: 15890675
15.  Genome-Wide Analysis of Binding Sites and Direct Target Genes of the Orphan Nuclear Receptor NR2F1/COUP-TFI 
PLoS ONE  2010;5(1):e8910.
Background
Identification of bona fide direct nuclear receptor gene targets has been challenging but essential for understanding regulation of organismal physiological processes.
Results
We describe a methodology to identify transcription factor binding sites and target genes in vivo by intersecting microarray data, computational binding site queries, and evolutionary conservation. We provide detailed experimental validation of each step and, as a proof of principle, utilize the methodology to identify novel direct targets of the orphan nuclear receptor NR2F1 (COUP-TFI). The first step involved validation of microarray gene expression profiles obtained from wild-type and COUP-TFI−/− inner ear tissues. Secondly, we developed a bioinformatic tool to search for COUP-TFI DNA binding sites in genomes, using a classification-type Hidden Markov Model trained with 49 published COUP-TF response elements. We next obtained a ranked list of candidate in vivo direct COUP-TFI targets by integrating the microarray and bioinformatics analyses according to the degree of binding site evolutionary conservation and microarray statistical significance. Lastly, as proof-of-concept, 5 specific genes were validated for direct regulation. For example, the fatty acid binding protein 7 (Fabp7) gene is a direct COUP-TFI target in vivo because: i) we identified 2 conserved COUP-TFI binding sites in the Fabp7 promoter; ii) Fapb7 transcript and protein levels are significantly reduced in COUP-TFI−/− tissues and in MEFs; iii) chromatin immunoprecipitation demonstrates that COUP-TFI is recruited to the Fabp7 promoter in vitro and in vivo and iv) it is associated with active chromatin having increased H3K9 acetylation and enrichment for CBP and SRC-1 binding in the newborn brain.
Conclusion
We have developed and validated a methodology to identify in vivo direct nuclear receptor target genes. This bioinformatics tool can be modified to scan for response elements of transcription factors, cis-regulatory modules, or any flexible DNA pattern.
doi:10.1371/journal.pone.0008910
PMCID: PMC2811727  PMID: 20111703
16.  COUP orphan receptors are negative regulators of retinoic acid response pathways. 
Molecular and Cellular Biology  1992;12(10):4666-4676.
The vitamin hormone retinoic acid (RA) regulates many complex biological programs. The hormonal signals are mediated at the level of transcription by multiple nuclear receptors. These receptors belong to the steroid/thyroid hormone receptor superfamily that also includes a large number of orphan receptors whose biological roles have not yet been determined. Although much has been learned in recent years about RA receptor (RAR) functions, little is known about how specific RA response programs are restricted to certain tissues and cell types during development and in the adult. It has been recently shown that RAR activities are regulated by retinoid X receptors (RXR) through heterodimer formation. In an effort to isolate and further characterize nuclear receptors that modulate RAR and/or RXR activities, we have screened cDNA libraries by using a RXR alpha cDNA probe. Two clones, COUP alpha and COUP beta, identical and closely related to the orphan receptor COUP-TF, were obtained. We show that COUP proteins dramatically inhibit retinoid receptor activities on certain response elements that are activated by RAR/RXR heterodimers or RXR homodimers. COUP alpha and -beta bind strongly to these response elements, including a palindromic thyroid hormone response element and a direct repeat RA response element as well as an RXR-specific response element. In addition, we found that the previously identified COUP-TF binding site in the ovalbumin gene functions in vitro as an RA response element that is repressed in the presence of COUP. Our data suggest that the COUP receptors are a novel class of RAR and RXR regulators that can restrict RA signaling to certain elements. The COUP orphan receptors may thus play an important role in cell- or tissue-specific repression of subsets of RA-sensitive programs during development and in the adult.
Images
PMCID: PMC360393  PMID: 1328857
17.  Proposed Role for COUP-TFII in Regulating Fetal Leydig Cell Steroidogenesis, Perturbation of Which Leads to Masculinization Disorders in Rodents 
PLoS ONE  2012;7(5):e37064.
Reproductive disorders that are common/increasing in prevalence in human males may arise because of deficient androgen production/action during a fetal ‘masculinization programming window’. We identify a potentially important role for Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) in Leydig cell (LC) steroidogenesis that may partly explain this. In rats, fetal LC size and intratesticular testosterone (ITT) increased ∼3-fold between e15.5-e21.5 which associated with a progressive decrease in the percentage of LC expressing COUP-TFII. Exposure of fetuses to dibutyl phthalate (DBP), which induces masculinization disorders, dose-dependently prevented the age-related decrease in LC COUP-TFII expression and the normal increases in LC size and ITT. We show that nuclear COUP-TFII expression in fetal rat LC relates inversely to LC expression of steroidogenic factor-1 (SF-1)-dependent genes (StAR, Cyp11a1, Cyp17a1) with overlapping binding sites for SF-1 and COUP-TFII in their promoter regions, but does not affect an SF-1 dependent LC gene (3β-HSD) without overlapping sites. We also show that once COUP-TFII expression in LC has switched off, it is re-induced by DBP exposure, coincident with suppression of ITT. Furthermore, other treatments that reduce fetal ITT in rats (dexamethasone, diethylstilbestrol (DES)) also maintain/induce LC nuclear expression of COUP-TFII. In contrast to rats, in mice DBP neither causes persistence of fetal LC COUP-TFII nor reduces ITT, whereas DES-exposure of mice maintains COUP-TFII expression in fetal LC and decreases ITT, as in rats. These findings suggest that lifting of repression by COUP-TFII may be an important mechanism that promotes increased testosterone production by fetal LC to drive masculinization. As we also show an age-related decline in expression of COUP-TFII in human fetal LC, this mechanism may also be functional in humans, and its susceptibility to disruption by environmental chemicals, stress and pregnancy hormones could explain the origin of some human male reproductive disorders.
doi:10.1371/journal.pone.0037064
PMCID: PMC3355148  PMID: 22615892
18.  Role of COUP-TFI during Retinoic Acid-Induced Differentiation of P19 Cells to Endodermal Cells 
Journal of cellular physiology  2013;228(4):791-800.
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.
doi:10.1002/jcp.24228
PMCID: PMC3657553  PMID: 23018522
COUP-TFI; COUP-TFII; retinoic acid; P19 cells
19.  Orphan Receptor COUP-TF Is Required for Induction of Retinoic Acid Receptor β, Growth Inhibition, and Apoptosis by Retinoic Acid in Cancer Cells 
Molecular and Cellular Biology  2000;20(3):957-970.
Retinoic acid receptor β (RARβ) plays a critical role in mediating the anticancer effects of retinoids. Expression of RARβ is highly induced by retinoic acid (RA) through a RA response element (βRARE) that is activated by heterodimers of RARs and retinoid X receptors (RXRs). However, RARβ induction is often lost in cancer cells despite expression of RARs and RXRs. In this study, we provide evidence that orphan receptor COUP-TF is required for induction of RARβ expression, growth inhibition, and apoptosis by RA in cancer cells. Expression of COUP-TF correlates with RARβ induction in a variety of cancer cell lines. In addition, stable expression of COUP-TF in COUP-TF-negative cancer cells restores induction of RARβ expression, growth inhibition, and apoptosis by RA, whereas inhibition of COUP-TF by expression of COUP-TF antisense RNA represses the RA effects. In a transient transfection assay, COUP-TF strongly induced transcriptional activity of the RARβ promoter in a RA- and RARα-dependent manner. By mutation analysis, we demonstrate that the effect of COUP-TF requires its binding to a DR-8 element present in the RARβ promoter. The binding of COUP-TF to the DR-8 element synergistically increases the RA-dependent RARα transactivation function by enhancing the interaction of RARα with its coactivator CREB binding protein. These results demonstrate that COUP-TF, by serving as an accessory protein for RARα to induce RARβ expression, plays a critical role in regulating the anticancer activities of retinoids.
PMCID: PMC85213  PMID: 10629053
20.  The spatial patterning of mouse cone opsin expression is regulated by BMP signaling through downstream effector COUP-TF nuclear receptors 
Cone photopigments, known as opsins, are pivotal elements and the first detection module employed in color vision. In mice, cone photoreceptors are distributed throughout the retina, and S- and M-opsins have unique expression patterns in the retina with a gradient along the dorsoventral axis; however, the mechanisms regulating the spatial patterning of cone opsin expression have not been well documented. The purpose of this study was to define the mechanisms regulating the spatial patterning of cone opsin expression. By analyzing knockouts for bone morphogenetic protein (BMP) signaling, we found an essential role for BMP in forming cone opsin expression patterns in the retina; however, BMP signaling is activated only transiently in the dorsal half of the retina during early retinal development. Thus, BMP is not likely to play a direct role in opsin gene expression, which starts at a later stage of retinal development. We identified the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) nuclear receptor as a link between BMP and opsin expression. BMP signaling is essential for the correct dorsoventral spatial expression of COUP-TFI and -TFII. Through gain- and loss-of-function analyses, we found that both COUP-TFI and -TFII are required to suppress S-opsin expression in the dorsal retina but that only COUP-TFI plays an essential role in suppressing M-opsin expression in the ventral retina. Based on these findings, we propose a new molecular cascade involving BMP and COUP-TFs that conveys dorsoventral information to direct the expression of cone opsins during retinal development.
doi:10.1523/JNEUROSCI.0951-09.2009
PMCID: PMC2791207  PMID: 19812316
BMP signal; mouse retina; nuclear receptor; S-opsin; M-opsin; photoreceptor
21.  Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development 
The Journal of Clinical Investigation  2010;120(5):1694-1707.
The lymphatic system plays a key role in tissue fluid homeostasis. Lymphatic dysfunction contributes to the pathogenesis of many human diseases, including lymphedema and tumor metastasis. However, the mechanisms regulating lymphangiogenesis remain largely unknown. Here, we show that COUP-TFII (also known as Nr2f2), an orphan member of the nuclear receptor superfamily, mediates both developmental and pathological lymphangiogenesis in mice. Conditional ablation of COUP-TFII at an early embryonic stage resulted in failed formation of pre-lymphatic ECs (pre-LECs) and lymphatic vessels. COUP-TFII deficiency at a late developmental stage resulted in loss of LEC identity, gain of blood EC fate, and impaired lymphatic vessel sprouting. siRNA-mediated downregulation of COUP-TFII in cultured primary human LECs demonstrated that the maintenance of lymphatic identity and VEGF-C–induced lymphangiogenic activity, including cell proliferation and migration, are COUP-TFII–dependent and cell-autonomous processes. COUP-TFII enhanced the pro-lymphangiogenic actions of VEGF-C, at least in part by directly stimulating expression of neuropilin-2, a coreceptor for VEGF-C. In addition, COUP-TFII inactivation in a mammary gland mouse tumor model resulted in inhibition of tumor lymphangiogenesis, suggesting that COUP-TFII also regulates neo-lymphangiogenesis in the adult. Thus, COUP-TFII is a critical factor that controls lymphangiogenesis in embryonic development and tumorigenesis in adults.
doi:10.1172/JCI40101
PMCID: PMC2860940  PMID: 20364082
22.  Chicken ovalbumin upstream promoter transcription factor (COUP-TF) dimers bind to different GGTCA response elements, allowing COUP-TF to repress hormonal induction of the vitamin D3, thyroid hormone, and retinoic acid receptors. 
Molecular and Cellular Biology  1992;12(9):4153-4163.
Alignment of natural chicken ovalbumin upstream promoter transcription factor (COUP-TF) response elements shows that, in addition to the predominant direct repeat of the GGTCA motif with a 2-bp spacing, there are other functional COUP elements with variations in the GGTCA orientation and spacing. We systematically analyzed the binding of in vitro-synthesized COUP-TFs and showed that COUP-TF is capable of binding to oligonucleotides containing both direct repeats and palindromes and with different spacings of the GGTCA repeats. Subsequently, we analyzed four possible mechanisms proposed to explain how COUP-TF could bind to these spatial variations of the GGTCA repeat. We demonstrated that the functional DNA-binding form of COUP-TF is a dimer which requires two GGTCA half-sites to bind DNA. We demonstrated that the COUP-TF dimer undergoes a remarkable structural adaptation to accommodate binding to these spatial variants of the GGTCA repeats. A functional consequence of the promiscuous DNA binding of COUP-TF is its ability to down-regulate hormonal induction of target gene expression by other members of the steroid-thyroid hormone receptor superfamily such as the vitamin D3, thyroid hormone, and retinoic acid receptors. Our data indicate that COUP-TF may have an important role in hormonal regulation of gene expression by these receptors.
Images
PMCID: PMC360317  PMID: 1324415
23.  Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) regulates growth and patterning of the postnatal mouse cerebellum 
Developmental biology  2008;326(2):378-391.
COUP-TFII (also known as Nr2f2), a member of the nuclear orphan receptor superfamily, is expressed in several regions of the central nervous system (CNS), including the ventral thalamus, hypothalamus, midbrain, pons, and spinal cord. To address the function of COUP-TFII in the CNS, we generated conditional COUP-TFII knockout mice using a tissue-specific NSE-Cre recombinase. Ablation of COUP-TFII in the brain resulted in malformation of the lobule VI in the cerebellum and a decrease in differentiation of cerebellar neurons and cerebellar growth. The decrease in cerebellar growth in NSECre/+/CIIF/F mice is due to reduced proliferation and increased apoptosis in granule cell precursors (GCPs). Additional studies demonstrated that insulin like growth factor 1 (IGF-1) expression was reduced in the cerebellum of NSECre/+/CIIF/F mice, thereby leading to decreased Akt1 and GSK-3β activities, and the reduced expression of mTOR. Using ChIP assays, we demonstrated that COUP-TFII was recruited to the promoter region of IGF-1 in a Sp1-dependent manner. In addition, dendritic branching of Purkinje cells was decreased in the mutant mice. Thus, our results indicate that COUP-TFII regulates growth and maturation of the mouse postnatal cerebellum through modulation of IGF-1 expression.
doi:10.1016/j.ydbio.2008.11.001
PMCID: PMC2654226  PMID: 19041640
COUP-TFII; IGF-1; Akt1; mTOR; Cerebellum; Foliation; GSK-3β
24.  Multiple roles of COUP-TFII in cancer initiation and progression 
Journal of molecular endocrinology  2012;49(3):R135-R148.
COUP-TFII is an orphan nuclear receptor that acts as a transcriptional activator or repressor in a cell type-dependent manner. Best characterized for its role in the regulation of angiogenesis during mouse development, COUP-TFII also plays important roles in glucose metabolism and cancer. Expression of COUP-TFII is altered in various endocrine conditions. Cell type-specific functions and the regulation of COUP-TFII expression result in its varying physiological and pathological actions in diverse systems. Evidence will be reviewed for oncogenic and tumor suppressive functions of COUP-TFII, with roles in angiogenesis, metastasis, steroidogenesis, and endocrine sensitivity of breast cancer described. The applicability of current data to our understanding of the role of COUP-TFII in cancer will be discussed.
doi:10.1530/JME-12-0144
PMCID: PMC3469769  PMID: 22966133
COUP-TFII; cancer; nuclear receptor
25.  Molecular mechanisms of COUP-TF-mediated transcriptional repression: evidence for transrepression and active repression. 
Molecular and Cellular Biology  1996;16(5):2332-2340.
COUP-TF, an orphan member of the nuclear receptor superfamily, has been proposed to play a key role in regulating organogenesis, neurogenesis, and cellular differentiation during embryonic development. Since heterodimierization is a common theme within the nuclear receptor superfamily and has been demonstrated to modulate transcriptional properties of heterodimeric partners via allosteric interactions, we have devised a strategy to examine the silencing function of COUP-TF in a heterodimeric context. We find that the intrinsic active repression function of COUP-TF is not affected by heterodimerization. Moreover, COUP-TF can transrepress the ligand-dependent activation of its heterodimeric partners without its own DNA binding site. Using receptor deletion mutants in transfection assays, we show that the region necessary for COUP-TF silencing function is not sufficient for its transrepression activity. Furthermore, our studies indicate that in addition to its active repression function, COUP-TF can repress several different types of activator-dependent transactivation. However, this active repression function of COUP-TF may be differentially regulated by some other activator(s). These studies provide new insights into the molecular mechanism(s) of COUP-TF-mediated repression.
PMCID: PMC231221  PMID: 8628300

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