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PubMed2004

Gene silencing by nuclear orphan receptors.

Nuclear orphan receptors represent a large and diverse subgroup in the nuclear receptor superfamily. Although putative ligands for these orphan members remain to be identified, some of these receptors possess intrinsic activating, inhibitory, or dual regulatory functions in development, differentiation, homeostasis, and reproduction. In particular, gene-silencing events elicited by chicken ovalbumin upstream promoter-transcription factors (COUP-TFs); dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1); germ cell nuclear factor (GCNF); short heterodimer partner (SHP); and testicular receptors 2 and 4 (TR2 and TR4) are among the best characterized. These orphan receptors are critical in controlling basal activities or hormonal responsiveness of numerous target genes. They employ multiple and distinct mechanisms to mediate target gene repression. Complex cross-talk exists between these orphan receptors at their cognate DNA binding elements and an array of steroid?nonsteroid hormone receptors, other transcriptional activators, coactivators and corepressors, histone modification enzyme complexes, and components of basal transcriptional components. Therefore, perturbation induced by these orphan receptors at multiple levels, including DNA binding activities, receptor homo- or heterodimerization, recruitment of cofactor proteins, communication with general transcriptional machinery, and changes at histone acetylation status and chromatin structures, may contribute to silencing of target gene expression in a specific promoter or cell-type context. Moreover, the findings derived from gene-targeting studies have demonstrated the significance of these orphan receptors' function in physiologic settings. Thus, COUP-TFs, DAX-1, GCNF, SHP, and TR2 and 4 are known to be required for multiple physiologic and biologic functions, including neurogenesis and development of the heart and vascular system steroidogenesis and sex determination, gametogenesis and embryonic development, and cholesterol?lipid homeostasis.

Animals↗

PubMed2000

Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process.

Steroidogenic factor-1 (SF-1, NR5A1a) is a member of the NR5A nuclear receptor subfamily and has been implicated as a key transcriptional regulator of all ovarian steroidogenic genes in vitro. To establish links between the expression of SF-1 and that of the steroidogenic genes in vivo, the objectives of this study were to clone equine SF-1 and examine the regulation of its messenger RNA (mRNA) in follicular cells during human CG (hCG)-induced ovulation. The equine SF-1 primary transcript was cloned by a combination of RT-PCR techniques. Results showed that the transcript was composed of a 5'-untranslated region (UTR) of 161 bp, an open reading frame (ORF) of 1386 bp that encodes a highly-conserved 461-amino acid protein, and a 3'-UTR of 518 bp. The cloning of SF-1 also led to the unexpected and serendipitous isolation of the highly-related orphan nuclear receptor NR5A2, which was shown to include a 5'-UTR of 243 bp, an ORF of 1488 bp, and a 3'-UTR of 1358 bp. The NR5A2 ORF encodes a 495-amino acid protein that is 60% identical to SF-1, including 99%-similar DNA-binding domains. Northern blot analysis revealed that SF-1 and NR5A2 were expressed in all major steroidogenic tissues, with the exception that NR5A2 was not present in the adrenal. Interestingly, NR5A2 was found to be, by far, the major NR5A subfamily member expressed in the preovulatory follicle and the corpus luteum. Using a semiquantitative RT-PCR/Southern blotting approach, the regulation of SF-1 and NR5A2 mRNAs in vivo was studied in equine follicular cells obtained from preovulatory follicles isolated between 0 and 39 h post hCG. Results showed that the theca interna was the predominant site of SF-1 mRNA expression in the follicle, and that hCG caused a significant decrease in SF-1 levels between 12-39 h in theca interna and between 24-39 h post hCG in granulosa cells (P < 0.05). In contrast, the granulosa cell layer was the predominant, if not the sole, site of NR5A2 mRNA expression in the follicle. Importantly, NR5A2 was much more highly expressed in granulosa cells than SF-1. The administration of hCG caused a significant decrease in NR5A2 transcripts in granulosa cells at 30, 36, and 39 h post hCG (P < 0.05). Thus, this study is the first to report the concomitant regulation of SF-1 in theca interna and granulosa cells throughout the ovulation/luteinization process, and to demonstrate the novel expression and hormonal regulation of NR5A2 in ovarian cells. Based on the marked expression of NR5A2 in equine granulosa and luteal cells and on mounting evidence of a functional redundancy between SF-1 and NR5A2 in other species, it is proposed that NR5A2 may play a key role in the regulation of gonadal steroidogenic gene expression.

Amino Acid Sequence↗

PubMed2004

The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation.

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1) (NROB1) is an atypical member of the nuclear receptor family, which lacks the classical zinc finger DNA binding domain and acts as a coregulator of a number of nuclear receptors. In this study, we have found that DAX-1 is a novel coregulator of the orphan nuclear receptor Nur77 (NR4A1). We demonstrate that DAX-1 represses the Nur77 transactivation by transient transfection assays. Specific interaction between Nur77 and DAX-1 was detected by coimmunoprecipitation, yeast two-hybrid, and glutathione-S-transferase pull-down assays. The ligand binding domain of DAX-1 and the activation function-2 domain of Nur77 were determined as the direct interaction domains between DAX-1 and Nur77. In vitro competition binding assay showed that DAX-1 repressed Nur77 transactivation through the competition with steroid receptor coactivator-1 for the binding of Nur77. Moreover, DAX-1 repressed Nur77- and LH-dependent increase of cytochrome P450 protein 17 promoter activity in transient transfection assays. Furthermore, Nur77-mediated transactivation was significantly increased by down-regulation of DAX-1 expression with DAX-1 small interfering RNA in testicular Leydig cell line, K28. LH treatment induced a transient increase in Nur77 mRNA, whereas LH repressed DAX-1 expression in a time- and dose-dependent manner in K28 cells. In addition, immunohistochemical analysis showed the expression of Nur77 in mouse testicular Leydig cells. These results suggest that DAX-1 acts as a novel coregulator of the orphan nuclear receptor Nur77, and that the DAX-1 may play a key role in the regulation of Nur77-mediated steroidogenesis in testicular Leydig cells.

Animals↗

PubMed2003

Molecular cloning and characterization of chicken orphan nuclear receptor cTR2.

Orphan nuclear receptors belong to the nuclear receptor superfamily of liganded transcription factors, whose ligands either do not exist or remain to be identified. We report here the cloning and characterization of the chicken orphan nuclear receptor, cTR2 (chicken testicular receptor 2). The cTR2 gene encodes a protein of 569 amino acids which shows approximately 72% overall identity with TR2 (NR2C1) and 95% identity in the DNA-binding domain (DBD). The cTR2 gene is expressed in almost all adult tissues and embryonic stages examined unlike its mammalian relative TR2, which is specifically expressed in testis. Electrophoretic mobility shift assays demonstrate that cTR2 binds the canonical direct repeat DNA recognition sequences spaced by one, four, and five nucleotides (DR1, DR4, and DR5), and in consistence with the results with canonical DNA-binding sequences, cTR2 forms specific DNA-protein complex with chicken phenobarbital response elements containing DR4 motifs. Both in vitro and in vivo interaction studies demonstrate that cTR2 forms homodimer. Moreover, transient transfection studies reveal its capability to transactivate canonical DR1, DR4, and DR5 sequences and the constitutive activity of cTR2 is mapped to the N-terminal region of this orphan receptor. Finally, cTR2 represses transactivation of estrogen receptor in a dose-dependent manner.

Amino Acid Sequence↗

PubMed1999

Identification of a novel DNA binding site for nuclear orphan receptor OR1.

The nuclear orphan receptor OR1 has been shown to bind as a heterodimer with retinoid X receptor (RXR) to direct repeat 4 (DR4) response elements. It remained unclear, however, whether this represents the only or the optimal binding site for this receptor. Therefore, we performed a DNA binding site selection assay that allows the identification of novel DNA binding sites for OR1 in an unbiased manner. While OR1 alone was not able to select a specific sequence from the pool of oligonucleotides, the OR1/RXR heterodimer selected a highly conserved DR1 element, termed DR1s, with two AGGTCA motifs spaced by one adenosine. The functional activity of the consensus binding site was verified in transient transfection assays and corroborated by in vitro studies. Based on the sequence of the consensus DR1s, we located putative natural binding sites in the 5'-promoter flanking regions of the rat S14 gene and the rat cholecystokinin type A receptor gene. Furthermore, we could show that although the OR1/RXR heterodimer has a distinct binding orientation on a DR4 element, it is able to bind in both orientations to the DR1s element. The OR1 paralog LXRalpha does not bind as a heterodimer with RXR to the DR1s element, indicating that these receptors, despite their homology, are involved in the regulation of different sets of genes.

Animals↗

PubMed1996

Retinoic acid receptors and nuclear orphan receptors in the development of Xenopus laevis.

Nuclear hormone receptors are ligand-activated transcription factors that regulate the expression of target genes by binding to hormone responsive elements (HRE) in their 5' upstream region. Retinoids which are known for their teratogenicity and which have a potential role in the specification of the anteroposterior axis of vertebrate embryos regulate transcription via a hormone-like mechanism by activating nuclear retinoic acid receptors, designated RAR and RXR. Of the several isoforms of RAR found in embryos of Xenopus laevis, xRAR gamma 2 appears to be the most abundant. During the early retinoic acid-sensitive period of development, the total amount of xRAR gamma 2 transcript and protein is increased and a highly specific pattern of expression emerges. During neurulation, the receptor is predominantly found in the dorsal posterior region, in the head endomesoderm, and in the rostral hindbrain. The dependence of this pattern on mesoderm induction and on neural induction is discussed. Contrasting with the elaborate pattern of xRAR gamma 2, the FTZ-F1-related nuclear orphan receptors (xFF1rA/B) are ubiquitous nuclear proteins in Xenopus embryos, as are the peroxisome proliferator-activated receptors xPPAR alpha and beta. PPARs are activated by polyunsaturated fatty acids and regulate the synthesis of enzymes involved in lipid metabolism. Later in development, the isoforms xPPAR alpha, beta, and gamma attain different tissue specificities.

Animals↗

PubMed1999

Hormone-independent transcriptional activation and coactivator binding by novel orphan nuclear receptor ERR3.

Orphan nuclear receptors share sequence homology with members of the nuclear receptor superfamily, but ligands are unknown or unnecessary. A novel orphan receptor, estrogen receptor-related protein 3 (ERR3), was identified by yeast two-hybrid screening, using the transcriptional coactivator glucocorticoid receptor interacting protein 1 (GRIP1) as bait. The putative full-length mouse ERR3 contains 458 amino acids and is closely related to two known orphan receptors ERR1 and ERR2. All the ERR family members share an almost identical DNA-binding domain, which has 68% amino acid identity with that of estrogen receptor. ERR3 bound specifically to an estrogen response element and activated reporter genes controlled by estrogen response elements, both in yeast and in mammalian cells, in the absence of any added ligand. A conserved AF-2 activation domain located in the hormone-binding domain of ERR3 was primarily responsible for transcriptional activation. The ERR3 AF-2 domain bound GRIP1 in a ligand-independent manner both in vitro and in vivo, through the LXXLL motifs of GRIP1, and GRIP1 functioned as a transcriptional coactivator for ERR3 in both yeast and mammalian cells. Expression of ERR3 in adult mouse was restricted; highest expression was observed in heart, kidney, and brain. In the mouse embryo no expression was observed at day 7, and highest expression occurred around the 11-15 day stages. Although ERR3 is much more closely related to ERR2 than to ERR1, the expression pattern for ERR3 was similar to that of ERR1 and distinct from that for ERR2, suggesting a unique role for ERR3 in development.

Amino Acid Sequence↗

PubMed1995

Functional interactions between retinoic acid receptor-related orphan nuclear receptor (ROR alpha) and the retinoic acid receptors in the regulation of the gamma F-crystallin promoter.

We have previously demonstrated that an everted repeat of the hexamer PuGGTCA located within the gamma F-crystallin promoter mediates activation of the murine gamma F-crystallin gene by retinoic acid and thyroid hormone receptors. Here, we show that the recently identified retinoic acid receptor-related orphan nuclear receptor (ROR alpha) is expressed in the murine lens and activates the gamma F-crystallin promoter. In contrast to the retinoic acid and thyroid hormone receptors, activation of the gamma F-crystallin promoter by ROR alpha requires binding to the single 3' half-site and spacer sequences of gamma F-crystallin hormone response element (gamma F-HRE). We further demonstrate that ROR alpha-dependent activation is repressed by the competitive binding of retinoic acid receptor/retinoid X receptor heterodimers to the gamma F-HRE in the absence of all-trans-retinoic acid. These studies suggest that the interplay of retinoid receptors and ROR alpha on the gamma F-HRE may constitute an important mechanism regulating gamma F-crystallin gene expression in the murine lens.

Animals↗

PubMed2005

An autoregulatory loop controlling orphan nuclear receptor DAX-1 gene expression by orphan nuclear receptor ERRgamma.

The estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the nuclear receptor superfamily that activates transcription in the absence of ligand. However, the detailed mechanism of gene regulation by ERRgamma is not fully understood. In this study we have found that the orphan nuclear receptor ERRgamma activates the DAX-1 promoter, which, in turn, represses transactivation by ERRgamma. Serial deletions of mouse DAX-1 (mDAX-1) gene promoter have revealed that the region responding to ERRgamma is located between -129 and -121 bp and -334 and -326 bp. Gel shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated that ERRgamma binds directly to the mDAX-1 promoter. Site-directed mutagenesis results demonstrated that ERRE1 (-129 to -121 bp) is more important than ERRE2 (-334 to -326 bp) which is not conserved in the human DAX-1 promoter. In addition, adenovirus-mediated overexpression of ERRgamma induced DAX-1 gene expression in MCF-7 breast cancer cells that co-expressed ERRgamma and DAX-1. Moreover, yeast two-hybrid and glutathione S-transferase (GST)-pull down assays demonstrated that DAX-1 physically interacted with ERRgamma and inhibited ERRgamma transactivation, and that this interaction was dependent on the AF-2 domain of ERRgamma. In addition, in vitro competition assays showed that DAX-1 inhibited PGC-1alpha mediated ERRgamma transactivation, via competition between these two factors for the AF-2 binding domain. We thus propose a novel autoregulatory loop that controls DAX-1 gene expression by ERRgamma.

Animals↗

PubMed2002

Defining requirements for heterodimerization between the retinoid X receptor and the orphan nuclear receptor Nurr1.

Nurr1, an orphan nuclear receptor mainly expressed in the central nervous system, is essential for the development of the midbrain dopaminergic neurons. Nurr1 binds DNA as a monomer and exhibits constitutive transcriptional activity. Nurr1 can also regulate transcription as a heterodimer with the retinoid X receptor (RXR) and activate transcription in response to RXR ligands. However, the specific physiological roles of Nurr1 monomers and RXR-Nurr1 heterodimers remain to be elucidated. The aim of this study was to define structural requirements for RXR-Nurr1 heterodimerization. Several amino acid substitutions were introduced in both Nurr1 and RXR in the I-box, a region previously shown to be important for nuclear receptor dimerization. Single amino acid substitutions introduced in either Nurr1 or RXR abolished heterodimerization. Importantly, heterodimerization-deficient Nurr1 mutants exhibited normal activities as monomers. Thus, by introducing specific amino acid substitutions in Nurr1, monomeric and heterodimeric properties of Nurr1 can be distinguished. Interestingly, substitutions in the RXR I-box differentially affected heterodimerization with Nurr1, retinoic acid receptor, thyroid hormone receptor, and constitutive androstane receptor demonstrating that the dimerization interfaces in these different heterodimers are functionally unique. Furthermore, heterodimerization between RXR and Nurr1 had a profound influence on the constitutive activity of Nurr1, which was diminished as a result of RXR interaction. In conclusion, our data show unique structural and functional properties of RXR-Nurr1 heterodimers and also demonstrate that specific mutations in Nurr1 can abolish heterodimerization without affecting other essential functions.

Amino Acid Sequence↗

PubMed2001

Differential regulation of the orphan nuclear receptor small heterodimer partner (SHP) gene promoter by orphan nuclear receptor ERR isoforms.

The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) interacts with a wide array of nuclear receptors and represses their transcriptional activity. SHP expression is regulated by several other members of the nuclear receptor superfamily, including the orphan receptors SF-1 and LRH-1, and the bile acid receptor FXR. We have found that the SHP promoter is also activated by the estrogen receptor-related receptor gamma (ERRgamma) but not the related ERRalpha and ERRbeta isoforms. SHP and ERRgamma mRNAs are coexpressed in several tissues, including pancreas, kidney, and heart, confirming the potential relevance of this transactivation. ERRgamma transactivation is dependent on only one of five previously characterized DNA-binding sites for SF-1, and this element differs from previously reported ERR response elements. However, treatment with the histone deacetylase inhibitor trichostatin A significantly increased ERRalpha and ERRbeta activity on this element indicating that the lack of activity of ERRalpha and -beta may depend on their association with co-repressor in vivo. Furthermore, using protease sensitivity assays on DNA bound receptors it was demonstrated that DNA sequence of different response elements may cause allosteric modulation of ERR proteins, which in turn may be responsible for the differential activities of these receptors on different response elements. SHP inhibits ERRgamma transactivation and physically interacts with all three members of ERR subfamily, as demonstrated by both yeast two-hybrid and biochemical assays. As with other SHP targets, this interaction is dependent on the AF-2 coactivator-binding site of ERRgamma and the previously described N-terminal receptor interaction domain of SHP. Several recently described SHP mutations associated with moderate obesity in humans block the inhibition of ERRgamma activity. Overall, these results identify a new autoregulatory loop controlling SHP gene expression and significantly extend the potential functional roles of the three ERRs.

Base Sequence↗

PubMed1997

Multiple orphan nuclear receptors converge to regulate rat P450c17 gene transcription: novel mechanisms for orphan nuclear receptor action.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays a key role in regulating the expression of the rat P450c17 gene in testicular Leydig and in adrenocortical cells. Other DNA sequences, not bound by SF-1, are also involved in transcriptional regulation of the rat P450c17 gene in both cell types. The region from -447/-399 or from -447/-419 increased both basal and cAMP-induced transcription, and the region from -418/-399 increased basal transcription to a greater extent than the intact -447/-399 DNA. The -447/-399 DNA sequence contains three imperfect copies of the orphan nuclear receptor-binding motif, AGGTCA, and at least three known orphan nuclear receptors, chicken ovalbumin upstream promoter transcription factor (COUP-TF), SF-1, and an early response gene induced by nerve growth factor (NGFI-B), bind to -447/-399 DNA. The AGGTCA triad is bound by one set of nuclear proteins when these three elements are colinear and is bound by a different set of proteins when these elements are separated. When the elements are separated, COUP-TF no longer binds, and the region -418/-399 is bound by a protein that greatly stimulates basal transcription. The region -447/-419 is bound by two different proteins that mediate both basal and cAMP-stimulated transcription. We call the protein binding to -418/-399 steroidogenic factor inducer of transcription-1 (StF-IT-1), and one of the proteins binding to -447/-419, StF-IT-2. SF-1 binds to a second AGGTCA element in the -447/-419 region. StF-IT-1 and StF-IT-2 are both found in Leydig and adrenal cells, and transcriptional regulation is similar in both cell types. SF-1 and NGF-IB may increase transcription by displacing COUP-TF (a transcriptional repressor) because these proteins share DNA-binding domains. However, neither SF-1 nor NGF-IB alone, binding as monomers, increases transcription. Rather, these proteins must interact with another DNA-binding protein, e.g. StF-IT-2, to increase transcription. StF-IT-2 also requires interaction with SF-1 (or NGF-IB) bound to DNA and cannot increase transcription by itself. This mechanism of action is different from the mechanism by which SF-1 regulates transcription from the -84/-55 region of the rat P450c17 gene. Thus, we have defined a novel mechanism of action for orphan nuclear receptors that bind to DNA as monomers.

Animals↗

PubMed2000

Constitutive activation of retinoic acid receptor beta2 promoter by orphan nuclear receptor TR2.

The orphan nuclear receptor TR2 functions as a constitutive activator for the endogenous retinoic acid receptor beta2 (RAR(beta2)) gene expression in P19 embryonal carcinoma cells and for reporters driven by the RAR(beta2) promoter in COS-1 cells. The activation of RAR(beta2) by TR2 is mediated by the direct repeat-5 (DR5) element located in the RAR(beta2) promoter. Furthermore, cAMP exerts an enhancing effect on the activation of RAR(beta2) by TR2, which is mediated by the cAMP response element located in the 5'-flanking region of the DR5. The constitutive activation function-1 (AF-1) of TR2 is mapped to amino acid residues 10-30 in its N-terminal A segment. A direct molecular interaction occurs between CREMtau and TR2, detected by co-immunoprecipitation, which is mediated by the N-terminal AB segment of TR2. In gel mobility shift assays, TR2 competes with P19 nuclear factor binding to the RAR(beta2) promoter, and TR2 and CREMtau bind simultaneously to this DNA fragment. The role of TR2 in the early events of RA signaling process is discussed.

Animals↗

PubMed2003

Synergistic activation of the human orphan nuclear receptor SHP gene promoter by basic helix-loop-helix protein E2A and orphan nuclear receptor SF-1.

The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) is an unusual orphan nuclear receptor that lacks a conventional DNA-binding domain and acts as a modulator of transcriptional activities of a number of nuclear receptors. We have previously reported that the orphan nuclear receptor ERRgamma activates the SHP promoter. In this study, we have found that basic helix-loop-helix (bHLH) transcription factors, the E2A proteins (E47, E12 and E2/5), activated the human but not the mouse SHP promoter. In contrast, the tissue-specific E47 heterodimer partner BETA2 repressed the E47- mediated transactivation of the human SHP (hSHP) promoter. Using serial deletions and E-box mutant constructs of the hSHP promoter, we identified two E-boxes (E6 and E7) as E47-responsive E-boxes, which are not conserved in the mouse SHP promoter. Moreover, gel shift, chromatin immunoprecipitation (ChIP) and northern blot assays demonstrated that E47 directly binds to the hSHP promoter in vivo and in vitro and that Id proteins inhibited E47 binding to the hSHP promoter. Finally, we found that E47 and steroidogenic factor 1 (SF-1), a regulator of the SHP promoter, synergistically activate the human but not the mouse SHP promoter. Our findings suggest that the E2A proteins differentially regulate the human and mouse SHP promoters and cooperate with orphan nuclear receptor SF-1 for transcriptional activation of the hSHP promoter.

Animals↗

PubMed1999

Identification of nuclear orphan receptors as regulators of expression of a neurotransmitter receptor gene.

Nuclear orphan receptors are known to be important mediators of neurogenesis, but the target genes of these transcription factors in the vertebrate nervous system remain largely undefined. We have previously shown that a 500-base pair fragment in the first intron of the GRIK5 gene, which encodes the kainate-preferring glutamate receptor subunit KA2, down-regulates gene expression. In our present studies, mutation of an 11-base pair element within this fragment resulted in a loss of nuclear protein binding and reverses negative regulation by the intron. Using yeast one-hybrid screening, we have identified intron-binding proteins from rat brain as COUP-TFI, EAR2, and NURR1. Gel shift studies with postnatal day 2 rat brain extract indicate the presence of COUP-TFs, EAR2, and NURR1 in the DNA-protein complex. Competition assays with GRIK5-binding site mutations show that the recombinant clones exhibit differential binding characteristics and suggest that the DNA-protein complex from postnatal day 2 rat brain may consist primarily of EAR2. The DNA binding activity was also observed to be enriched in rat neural tissue and developmentally regulated. Co-transfection assays showed that recombinant nuclear orphan receptors function as transcriptional repressors in both CV1 cells and rat CG4 oligodendrocyte cells. Direct interaction of the orphan receptors with and relief of repression by TFIIB indicate likely role(s) in active and/or transrepression. Our findings are thus consistent with the notion that multiple nuclear orphan receptors can regulate the transcription of a widely expressed neurotransmitter receptor gene by binding a common element in an intron and directly modulating the activity of the transcription machinery.

Animals↗

PubMed2003

Identification of histone deacetylase-3 domains that interact with the orphan nuclear receptor TR2.

The orphan nuclear receptor TR2 interacts directly with histone deacetylase HDAC3 and HDAC4. We now report that two domains of HDAC3 are involved in its interaction with TR2. GST pull-down assays show that both the N-terminal (residues 1-135) and the C-terminal (residues 210-428) segments of HDAC3 directly interact with TR2. The interaction is also demonstrated in coimmunoprecipitation experiments. The two TR2-binding sites of HDAC3 compete with each other for binding to TR2. The two receptor-interacting domains (RIDs) of HDAC3 were further dissected and mapped to amino acid residues 1-70 and 270-320. In vivo studies demonstrate that HDAC3 and TR2 can form a complex on the TR2 DNA target and this complex exhibits histone deacetylase activity. These data identify two RIDs of HDAC3 and the biological activity of the complex formed by TR2 and HDAC3 on the TR2 DNA target.

Animals↗

PubMed2004

Growth retardation and abnormal maternal behavior in mice lacking testicular orphan nuclear receptor 4.

Testicular orphan nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily for which a ligand has not yet been found. In vitro data obtained from various cell lines suggest that TR4 functions as a master regulator to modulate many signaling pathways, yet the in vivo physiological roles of TR4 remain unclear. Here, we report the generation of mice lacking TR4 by means of targeted gene disruption (TR4(-/-)). The number of TR4(-/-) pups generated by the mating of TR4(+/-) mice is well under that predicted by the normal Mendelian ratio, and TR4(-/-) mice demonstrate high rates of early postnatal mortality, as well as significant growth retardation. Additionally, TR4(-/-) females show defects in reproduction and maternal behavior, with pups of TR4(-/-) dams dying soon after birth with no indication of milk intake. These results provide in vivo evidence that TR4 plays important roles in growth, embryonic and early postnatal pup survival, female reproductive function, and maternal behavior.

Animals↗

PubMed2006

Multiple signaling pathways regulate the transcriptional activity of the orphan nuclear receptor NURR1.

The orphan nuclear receptor nurr1 (NR4A2) is an essential transcription factor for the acquisition and maintenance of the phenotype of dopamine (DA)-synthesizing neurons in the mesencephalon. Although structurally related to ligand-regulated nuclear receptors, nurr1 is functionally atypical due to its inability to bind a cognate ligand and to activate transcription following canonical nuclear receptor (NR) rules. Importantly, the physiological stimuli that activate this NR and the signaling proteins that regulate its transcriptional activity in mesencephalic neurons are unknown. We used an affinity chromatography approach and CSM14.1 cells of mesencephalic origin to isolate and identify several proteins that interact directly with nurr1 and regulate its transcriptional activity. Notably, we demonstrate that the mitogen-activated protein kinases, ERK2 and ERK5, elevate, whereas LIM Kinase 1 inhibits nurr1 transcriptional activity. Furthermore, nurr1 recruits ERK5 to a NBRE-containing promoter and is a potential substrate for this kinase. We have identified amino acids in the A/B domain of nurr1 important for mediating the ERK5 activating effects on nurr1 transcriptional activity. Our results suggest that nurr1 acts as a point of convergence for multiple signaling pathways that likely play a critical role in differentiation and phenotypic expression of dopaminergic (DAergic) neurons.

Animals↗