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Identification of Human TR2 Orphan Receptor Response Element in the Transcriptional Initiation Site of the Simian Virus 40 Major Late Promoter
Abstract
A DNA response element (TR2RE-SV40) for the TR2 orphan receptor, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5'-GTTAAGGTTCGTAGGTCATGGA-3'). Electrophoretic mobility shift assay, using in vitro translated TR2 orphan receptor with a molecular mass of 67 kilodaltons, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using chloramphenicol acetyl-transferase assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the chimeric receptor AR/TR2/AR with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the androgen receptor. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the TR2 orphan receptor that may function as a repressor for the SV40 gene expression.
... The medium was changed to Dulbecco's modified Eagle's medium with 5% charcoal dextran-treated serum at least 1 h before transfection. The cells were transfected using a modified calcium phosphate precipitation method (22). To normalize the transfection efficiency, the -galactosidase expression vector was co-transfected. ...
... This is true for both synthetic and native T 3 REs as well as other nonclassical T 3 REs. DISCUSSION Like the TR2 orphan receptor, TR4 can also serve as a repressor in RAR/RXR-mediated gene induction and transcription from the major late promoter of the SV40ϩ55 gene (16,22,28). In the present study, we showed TR4 can also induced the transcriptional activity of genes containing a DR4 or DR4-like sequence. ...
... In Fig. 1, when we replaced the third G with C in DR4-HRE (mDR4), an excessive amount of unlabeled mDR4 could not abolish with the specific DNA-receptor complex completely. This suggests that the precise sequence of the core motif plays an essential role in TR4-mediated gene regulation, a result in agreement with our previous studies as the TR2 orphan receptor (22,28). In addition, the spacing of core binding motifs may also be a key factor in dictating specificity of transcriptional response to a transcriptional factor. ...
While the TR4 orphan receptor (TR4) is able to repress the expression of its target genes via its interaction with the direct
repeat 1-hormone response element (DR1-HRE) and DR2-HRE, we now report that TR4 can also induce the transcriptional activity
of the reporter gene containing a DR4-HRE via chloramphenicol acetyltransferase assay. Electrophoretic mobility shift assay
and Scatchard analysis reveal a strong binding affinity (dissociation constant = 2 nm) between TR4 and DR4-HRE. The induction mediated by TR4 was detected not only in the synthetic DR4-HRE but also in some genes,
such as rat α-myosin heavy-chain and S14 genes, containing the DR4 or DR4-like motif, which have been suggested to be the
response elements for a thyroid hormone receptor. Our data also demonstrate this TR4-mediated gene induction is TR4 dose-
and DR4 sequence-dependent. Together, our data suggest that DR4-HRE can be a positive regulatory element for TR4, which may
be able to induce the transcriptional activity of the genes containing such positive HREs.
... Our top candidate, NR2C1, belongs to a subtype of NRs known as orphan receptors for which the endogenous ligand (if any) has yet to be identified (Lee and Chang 1995). Originally named testicular receptor 2 (TR2) because it was first isolated from human testis and prostate (Chang and Kokontis 1988;Anderson et al. 2012), its expression in ES cells and in pluripotent cell culture lines indicates it plays a role in early embryonic development (Hu et al. 2002). ...
... Originally named testicular receptor 2 (TR2) because it was first isolated from human testis and prostate (Chang and Kokontis 1988;Anderson et al. 2012), its expression in ES cells and in pluripotent cell culture lines indicates it plays a role in early embryonic development (Hu et al. 2002). It is one of a handful of genes implicated in the regulation of the pluripotentiality of stem cell populations in the embryo, and in neural stem cells in particular (Lee and Chang 1995;Lee et al. 2002;Hu et al. 2002;Shyr et al. 2009). In addition, NR2C1 has been shown to regulate the expression of Oct4 and Nanog, two transcription factors essential for maintaining the pluripotentiality of embryonic stem cells (Pikarsky et al. 1994;Niwa et al. 2000;Boiani 2002). ...
Genes encoding nuclear receptors (NRs) are attractive as candidates for investigating the evolution of gene regulation because they have 1) a direct effect on gene expression, and 2) modulate many cellular processes that underlie development. We employed a three-phase investigation linking NR molecular evolution among primates with direct experimental assessment of NR function. Phase 1 was an analysis of NR domain evolution and the results were used to guide the design of Phase 2, a codon-model based survey for alterations of natural selection within the hominids. By using a series of reliability and robustness analyses we selected a single gene,NR2C1, as the best candidate for experimental assessment. We carried out assays to determine if changes between the ancestral and extantNR2C1s could have impacted stem cell pluripotency (Phase 3). We evaluated human, chimpanzee, and ancestralNR2C1for transcriptional modulation ofOct4andNanog(key regulators of pluripotency and cell lineage commitment), promoter activity forPepck(a proxy for differentiation in numerous cell types), and average size of embryological stem cell colonies (a proxy for the self-renewal capacity of pluripotent cells). Results supported the signal for alteration of natural selection identified in Phase 2. We suggest that adaptive evolution of gene regulation has impacted several aspects of pluripotentiality within primates. Our study illustrates that the combination of targeted evolutionary surveys and experimental analysis is an effective strategy for investigating the evolution of gene regulation with respect to developmental phenotypes.
... By using a reporter containing a DR4-type hormone response element of the mouse cellular retinoic acid binding protein I (CRABP-I) gene, we have also shown that TR2, but not TR2-11-t, strongly repressed this reporter (Chinpaisal et al. 1997). In other promoter systems such as the Simian virus (SV) 40 promoter (Lee & Chang 1995) and the erythropoietin gene promoter (Lee et al. 1996b), TR2 also functions primarily as a transcriptional repressor (Lin et al. 1995). ...
The orphan nuclear receptor TR2 and its truncated isoform deleted in the ligand binding domain (LBD) were localized exclusively in the nuclei as revealed by two methods of detection. An anti-hemagglutinin (HA) antibody detected specific nuclear localization of HA-tagged receptors and the green fluorescent protei (GFP)-tagged receptor ere found to be distributed in the nuclei of living cells. By deletion analyses, the sequence responsible for targeting this receptor into the nucleus was defined. A stretch of 20 amino acid residues (KDCVINKHHRN RCQYCRLQR) within the second zinc-finger of this receptor is required for its nuclear localization and this signal is constitutively active. No nuclear localization signal was found in the N-terminus or the LBD. The GFP-tagged receptor remained biologically active, as evidenced by its repressive activity on the reporter that carried a binding site for this receptor, a direct repeat-5 (DR5). An electrophoretic mobility shift assay was performed to characterize the binding property of TR2 and its truncated isoform. TR2 bound to the DR5 as dimers whereas its truncated isoform bound as monomers.
... Polyacrylamide gel electrophoresis (PAGE) technique continues to play a major role in protein purification [1] and in protein/protein and protein/small ligand interaction studies [2]. The advancement in protein engineering and in peptide synthesis has enabled the synthesis of diverse forms of oligopeptides of medicinal interest [3,4]. ...
A simple and fast procedure that allows the separation of small (1–3 kDa) peptides on glycine-SDS gels is described. Peptides
were separated by glycine-SDS/PAGE as a result ofin situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented
here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamide mini
gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation
and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.
This chapter focuses on recent accomplishments and future directions in the nuclear orphan receptor field. The subsequent identification of ligands and activators for other orphan receptors has implicated the involvement of these novel regulators in a myriad of signaling pathways not previously known to involve nuclear receptors. Many of these orphan receptors are also associated with metabolic and inherited disorders and they may be ideal targets for the development of improved pharmacologic agents for therapeutic use. Nuclear receptors are classically defined as ligand-activated transcription factors that share several common structural features that allow for DNA binding and transcriptional activation. Members of the nuclear receptor superfamily fall into two distinct evolutionary and functional groups: the steroid hormone receptors and the nonsteroid hormone receptors. In addition to the successful cell-based assays that have been used, recent progress in the disciplines of X-ray crystallography and NMR have begun to provide insights on the mechanism of DNA and ligand binding, as well as transcriptional activation by the nuclear receptors. In addition to the successful cell-based assays that have been used, recent progress in the disciplines of X-ray crystallography and NMR also provide insights on the mechanism of DNA and ligand binding as well as transcriptional activation by the nuclear receptors.
Le virus d’Epstein-Barr (EBV) est un Herpesvirus humain appartenant à la sous-famille des γ-Herpesvirinae. L’expression des gènes d’EBV est régulée très finement, de manière séquentielle et débute par l’expression des gènes immédiats précoces dont les produits contrôlent l’expression des gènes précoces. Les produits de ces derniers permettent la réplication de l’ADN viral et la synthèse des gènes tardifs. Les mécanismes contrôlant l’expression des gènes immédiats précoces et précoces sont assez bien compris, à contrario de ceux qui contrôlent l’expression des gènes tardifs. En effet, il est connu que ces derniers ne sont exprimés qu’après réplication de l’ADN viral. Par ailleurs, les promoteurs des gènes tardifs présentent une structure différente des promoteurs de gènes immédiats précoces, précoces ou des gènes cellulaires. Ils sont essentiellement caractérisés par la présence d’une boîte TATT à la place de la TATA-box canonique. EBV a la particularité de coder pour une TBP-like, la protéine BcRF1 qui est essentielle mais pas suffisante pour l’expression des gènes viraux tardifs et qui se fixe spécifiquement sur la séquence TATT des promoteurs viraux tardifs. Notre objectif était principalement d’identifier l’ensemble des protéines virales nécessaires à l’expression des gènes tardifs d’EBV. Nous avons ainsi démontré qu’en plus de la protéine BcRF1, EBV code pour cinq autres protéines nécessaires à l’expression des gènes viraux tardifs : BFRF2, BGLF3, BVLF1, BDLF4 et BDLF3.5. Ces six protéines virales forment un complexe qui permet le recrutement de l’ARN polymérase II sur les promoteurs des gènes viraux tardifs. Nous avons nommé ce complexe vPIC (viral Pre-Initiation Complex), par analogie avec le complexe d’initiation de la transcription cellulaire formé autour de la TATA-Binding Protein (TBP). Le vPIC est conservé chez les herpesvirus des sous familles β et γ, mais est absent chez les herpesvirus de la sous famille des α. Par ailleurs, nos résultats suggèrent que le complexe vPIC interagit avec le complexe de réplication viral ce qui peut expliquer la liaison entre réplication de l’ADN viral et transcription des gènes viraux tardifs. Ces résultats permettent ainsi de mieux comprendre les mécanismes de régulation de l’expression des gènes tardifs d’EBV et serviront de base pour la recherche de nouveaux antiviraux.
The key expression of the simian virus 40 (SV40) major late promoter could be repressed by the human TR4 orphan receptor via
the +55 region of the SV40 major late promoter (nucleotide numbers 368-389, 5′-GTTAAGGTTCGTAGGTCATGGA-3′). Using the coupled
in vitro transcribed and translated TR4 orphan receptor with a molecular mass of 67.3 kilodaltons, electrophoretic mobility shift
assay showed specific binding with a dissociation constant of 1.09 nM between the TR4 orphan receptor and the SV40 +55 oligonucleotides. In addition, chloramphenicol acetyltransferase assay demonstrated that this SV40 +55 region can
function as a repressor via the TR4 orphan receptor, suppressing the transcriptional activities of both SV40 early and late
promoters. Together, our data suggest that the TR4 orphan receptor may play an important role for the suppression of the SV40
gene expression.
Nuclear Receptor subfamily 1, group H, member 4 (NR1H4) is a receptor for bile acids and has an important role in regulating energy metabolism in liver, muscle and adipose tissues in humans and animals. In this study, we cloned the full coding region of NR1H4 gene from porcine Longissimus dorsi by Rapid amplification of cDNA end (RACE). Results indicated that the open reading frame of NR1H4 covered 1461 bp encoding 486 amino acid residues and the deduced amino acid sequence was 91-94 % identical to that of Homo sapiens, Bos taurus, Macaca mulatta, Gorilla gorilla, and Ovis aries. Bioinformatic analysis indicated that NR1H4 contained 31 phosphorylation sites with 14 serine, 6 threonine and 11 tyrosine. One single nucleotide polymorphism (SNP) was detected by PCR-RFLP in 3' untranslated region of exon 9 (NR1H4) and the allele frequency analysis showed that A allele frequency was low among 396 pigs from five breeds. The NR1H4 mRNA expression pattern showed that NR1H4 gene was expressed highly in live and Longissimus dorsi. This work provided an important experimental basis for further research on mechanism of lipid metabolism and fat deposition in pigs.
Although many co-activators have been identified for various nuclear receptors, relatively fewer co-repressors have been isolated
and characterized. Here we report the identification of a novel testicular orphan nuclear receptor-4 (TR4)-associated protein
(TRA16) that is mainly localized in the nucleus of cells as a repressor to suppress TR4-mediated transactivation. The suppression
of TR4-mediated transactivation is selective because TRA16 shows only a slight influence on the transactivation of androgen
receptor, glucocorticoid receptor, and progesterone receptor. Sequence analysis shows that TRA16 is a novel gene with 139
amino acids in an open reading frame with a molecular mass of 16 kDa, which did not match any published gene sequences. Mammalian
two-hybrid system and co-immunoprecipitation assays both demonstrate that TRA16 can interact strongly with TR4. The electrophoretic
mobility shift assay suggests that TRA16 may suppress TR4-mediated transactivation via decreased binding between the TR4 protein
and the TR4 response element on the target gene(s). Furthermore, TRA16 can also block the interaction between TR4 and TR4
ligand-binding domain through interacting with TR4-DNA-binding and ligand-binding domains. These unique suppression mechanisms
suggest that TRA16 may function as a novel repressor to selectively suppress the TR4-mediated transactivation.
The cellular localization and expression of TR2 mRNA in rhesus monkey testis were investigated by the methods ofin situ and Northern hybridization using the digoxigenin labeledin vitro transcripted cRNA probe from a TR2 cDNA fragment template and α-32P-dCTP labeled cDNA probe from the same TR2 cDNA fragment. It is demonstrated that TR2 mRNA was localized specifically in
the germ cells, and was predominantly expressed in the meiotic more advanced cells. The expression level was various in different
seminiferous tubules, and there was stronger expression in the tubules where spermatogenesis was vigorously going on. The
expression level of TR2 mRNA was low in the immature rhesus monkey testis and increased dramatically in the adult and varied
with a seminiferous cycle. These data suggest for the first time that orphan receptor TR2 may have an important function in
regulating spermatogenesis at later stages of germ cell development in rhesus monkey.
The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains a negative regulatory element (NRE) which downregulates the rate of LTR-directed transcription and HIV-1 replication. Within the NRE is a GGTCA palindrome, which binds a possible member of the steroid/thyroid hormone receptor superfamily. Mutation of this site leads to an increase in LTR-directed transcriptional activity compared with the wild type, consistent with the element's being a functional part of the NRE. The palindrome contains significant identity to the chicken ovalbumin upstream promoter (COUP) element to which COUP transcription factors (COUP-TFs), members of the steroid/thyroid hormone receptor superfamily, bind. We demonstrate here that human COUP-TFs can bind specifically to this HIV-1 COUP-like element in a manner identical to binding to ovalbumin COUP. We show that the predominant COUP-TF family member synthesized in T cells is the 68-kDa form, which is likely to be responsible for any in vivo function of the HIV-1 COUP-like element in these cells. Finally, we have identified three HIV-1 variant strains that contain mutations in the HIV-1 COUP-like element which affect the binding affinity of COUP-TF for these variant COUP elements.
Several steroid hormones induce transcription of the mouse mammary tumor virus (MMTV) promoter, through an interaction of their respective receptors with the hormone responsive elements (HREs) in the long terminal repeat (LTR) region. The molecular mechanism underlying transcriptional activation is not known, but binding of nuclear factor I (NFI) to a site adjacent to the HRE appears to be required for efficient transcription of the MMTV promoter. In JEG-3 choriocarcinoma cells the MMTV promoter is transcribed inefficiently, even after transfection of the receptor cDNA and treatment with glucocorticoids or progestins. These cells contain low levels of NFI as cotransfection of NFI cDNA enhances MMTV transcription and this effect is inhibited by mutation of the NFI binding site. In DNA binding experiments with purified NFI from pig liver, the glucocorticoid and progesterone receptors do not co-operate but rather compete with NFI for binding to their respective sites on the LTR. Similar results are obtained with a functional recombinant NFI synthesized in vitro. Competition for DNA binding is probably due to steric hindrance as the DNase I footprints of the hormone receptors and NFI do overlap. These results suggest that, though NFI acts as a transcription factor on the MMTV promoter, transcriptional activation does not take place through a direct facilitation of DNA binding of NFI by steroid hormone receptors.
We describe the isolation, cloning, and structural characterization of cDNAs encoding TR2 receptor and TR3 receptor and the expression of the receptor proteins. The natural ligands for these nuclear receptor-like proteins are unknown. TR2 receptor exists in four variant forms which differ in the putative ligand-binding domain. The expression of TR2 receptor in rat ventral prostate and TR2 receptor and TR3 receptor in the human prostatic carcinoma cell line LNCaP is discussed.
Characterization of several thyroid hormone (T3), retinoic acid, and estrogen response elements has led to the identification of conserved DNA half-sites (core binding motifs). We present evidence that differences in both the relative orientation and spacing of these motifs within hormone response elements determine the distinct transcriptional responses of three members of the nuclear receptor superfamily. When separated by 3 bp, direct repeat, palindromic, and inverted palindromic arrangements of these motifs impart selective transcriptional responses to retinoic acid, estrogen, and T3 receptors, respectively. Varying the spacing between core motifs alters the specificity. Without spacing, a direct repeat of the core motif paradoxically configures the T3 receptor to confer transactivation in the absence of T3 and repression in its presence. Such an element occurs naturally in the mouse beta-thyrotropin promoter, physiologically under negative regulation by T3. The orientation and spacing of core binding motifs may thus function in concert as a code that accounts for the selective patterns of transcriptional responses of hormonally regulated promoters.
An in vivo selection system for isolating targets of DNA binding proteins in yeast was developed and used to identify the
DNA binding site for the NGFI-B protein, a member of the steroid-thyroid hormone receptor superfamily. The feasibility of
the technique was verified by selecting DNA fragments that contained binding sites for GCN4, a well-characterized yeast transcriptional
activator. The DNA binding domain of NGFI-B, expressed as part of a LexA-NGFI-B-GAL4 chimeric activator, was then used to
isolate a rat genomic DNA fragment that contained an NGFI-B binding site. The NGFI-B response element (NBRE) is similar to
but functionally distinct from elements recognized by the estrogen and thyroid hormone receptors and the hormone receptor-like
proteins COUP-TF, CF1, and H-2RIIBP. Cotransfection experiments in mammalian cells demonstrated that NGFI-B can activate transcription
from the NBRE with or without its putative ligand binding domain.
The molecular specificity of the receptors for steroid and thyroid hormones is achieved by their selective interaction with DNA binding sites referred to as hormone response elements (HREs). HREs can differ in primary nucleotide sequence as well as in the spacing of their dyadic half-sites. The target gene specificity of the glucocorticoid receptor can be converted to that of the estrogen receptor by changing three amino acids clustered in the first zinc finger. Remarkably, a single Gly to Glu change in this region produces a receptor that recognizes both glucocorticoid and estrogen response elements. Further replacement of five amino acids in the stem of the second zinc finger transforms the specificity to that of the thyroid hormone receptor. These findings localize structural determinants required for discrimination of HRE sequence and half-site spacing, respectively, and suggest a simple pathway for the coevolution of receptor DNA binding domains and hormone-responsive gene networks.
Previously we isolated a new group of cDNA clones from human testis cDNA libraries which might code for new steroid receptors. The cDNA and predicted amino acid sequences of two of these receptors, named TR2-5 and TR2-7 receptors, were determined. We report here the nucleotide and deduced amino acid structures of two other receptors that we named TR2-9 and TR2-11 receptors. The calculated MW of TR2-5 receptor, TR2-7 receptor, TR2-9 receptor and TR2-11 receptor are 52,982, 20,528, 50,849 and 67,223 respectively, which match well with the apparent MW of in vitro translated products. The 26 amino acids involved in the formation of "Zn-fingers" are conserved. The ligand-binding domain of TR2-9 receptor is 16 amino acids shorter and has 3 different amino acids compared with TR2-5 receptor. The TR2-11 receptor has a ligand-binding domain which is longer and quite different compared with the other TR2 receptors. The multiple ligand-binding domains of TR2 receptor could be the products of different genes or may be due to RNA splicing errors. So far, we have failed to find binding activity with any known steroid hormone; this promotes the possibility that an unidentified steroid hormone may be involved.