Thyroid Hormone Receptor DNA Binding Is Required for Both Positive and Negative Gene Regulation*

Nobuyuki ShibusawaDagger , Anthony N. Hollenberg§, and Fredric E. WondisfordDagger

From the Dagger  Section of Endocrinology, Department of Medicine, the University of Chicago, Chicago, Illinois 60637 and § Division of Endocrinology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Received for publication, July 19, 2002, and in revised form, October 22, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The beta  isoform of thyroid hormone receptor (TR-beta ) has a key role in the feedback regulation of the hypothalamic-pituitary-thyroid (H-P-T) axis. The mechanism of trans-repression of the hypothalamic thyrotropin-releasing hormone (TRH) and pituitary thyroid-stimulating hormone (TSH) subunit genes, however, remains poorly understood. A number of distinct mechanisms for TR-beta -mediated negative regulation by thyroid hormone have been proposed, including those that require and do not require DNA binding. To clarify the importance of DNA binding in negative regulation, we constructed a DNA-binding mutant of TR-beta in which two amino acids within the P box were altered (GSG for EGG) to resemble that found in the glucocorticoid receptor (GR). We termed this mutant GS125, and as expected, it displayed low binding affinities for positive and negative thyroid hormone-response element (pTRE and nTRE, respectively) in gel-mobility shift assays. In transient transfection assays, the GS125 mutant abolished transactivation on three classic pTREs (DR+4, LAP, and PAL) and all negatively regulated promoters in the H-P-T axis (TRH, TSH-beta , and TSH-alpha ). However, GS125 TR-beta bound to a composite TR/GR-response element and was fully functional on this hybrid TR/GR-response element. Moreover, the GS125 TR-beta mutant displayed normal interactions with transcriptional cofactors in mammalian two-hybrid assays. These data do not support a DNA-binding independent mechanism for thyroid hormone negative regulation in the H-P-T axis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The synthesis and secretion of thyroid hormones are exquisitely regulated by a negative feedback system that involves the hypothalamus, pituitary, and thyroid gland (the hypothalamic-pituitary-thyroid axis: H-P-T axis).1 The synthesis of thyrotropin-releasing hormone (TRH), produced in the paraventricular nucleus of the hypothalamus, and the alpha  and beta  subunits of thyrotropin (thyroid-stimulating hormone; TSH) in the anterior lobe of the pituitary is inhibited at the transcriptional level by thyroid hormone (T3) (1-4). Negative regulation of gene expression by T3 is an integral part of the function in the H-P-T axis, and these effects are mediated by the beta  isoform of the thyroid hormone receptors (TR-beta ). Of the beta  isoforms, TR-beta 2 is quantitatively and functionally more important than TR-beta 1 in regulating the hypothalamus and pituitary (5-8). The molecular mechanisms responsible for TR-mediated negative regulation of target genes are not well understood.

In the traditional model of TR action, TR regulates gene expression by binding to specific thyroid hormone-response elements (TRE) on target genes. A number of artificial and natural positive thyroid hormone-response elements (pTREs) have been described, and they can be classified based on the half-site arrangement in the element: DR+4, PAL, and LAP (LYS) (9-11). On target genes negatively regulated by T3, response elements (nTREs) have been described in the TRH and TSH-beta subunit genes (12-17). In these nTREs, TR bound as a monomer to widely spaced half-sites or in the case of TRH with retinoid X receptor (RXR) to an unusual element (13, 18-20). Mutagenesis studies (21, 22) suggest that TR DNA binding to the half-site is required for negative regulation. Activation of these nTRE in the absence of T3 is mediated by the N terminus of TR-beta 2 via a mechanism involving binding of coactivator proteins (23, 24).

In contrast, some investigators have suggested that TR DNA binding is not required for negative regulation. This mechanism has been proposed to explain negative regulation of the TSH-alpha promoter where an nTRE has not been clearly identified (25, 26). In this model, corepressors bind to TR in solution and activate gene expression; in the presence of T3, corepressors dissociate from the TR and return to the TSH-alpha promoter to repress gene expression. A major concern in this model, however, is how specificity of negative regulation is achieved in the absence of DNA binding. Thus, both on and off DNA binding mechanisms have been proposed to explain negative regulation by thyroid hormone.

TRs, like other members of the superfamily of nuclear hormone receptors, have a conserved protein structure including a central DNA binding domain (DBD) and a C-terminal ligand binding domain (27, 28). The DBD is composed of two zinc finger motifs in which the zinc ion is tetrahedrally coordinated by four cysteines (29, 30). C-terminal to each zinc finger is an amphipathic alpha -helix. The alpha -helix of the first finger motif is responsible for sequence-specific DNA recognition, whereas the alpha -helix following the second finger motif provides phosphate contacts to the DNA and stabilizes the structure (31). The central two nucleotides of the hexameric half-site consensus motif (AGNNCA) in a hormone-response element is determined by three receptor-specific amino acids in the DNA recognition alpha -helix, referred to as the P box (32). Nuclear receptors have been divided into two subfamilies according to the identity of their P box amino acid sequences (33). The subfamily of glucocorticoid receptor (GR) has a GSV (glycine-serine-valine) P box sequence and is able to bind to DNA elements containing the nucleotide sequence of AGAACA. The subfamily that includes the TRs has a more diverse P box sequence. TRs contain an EGG (glutamic acid-glycine-glycine) P box sequence and recognize DNA elements with the nucleotide sequence of AGGTCA (Fig. 1).


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Fig. 1.   Schematic diagram of the zinc finger region of the DBD of thyroid hormone receptor beta . The shaded circles indicate the P box amino acids. The boxed region outlines the DNA recognition alpha -helix. The GR and thyroid hormone receptor beta  P box sequence and their consensus DNA-binding half-sites are shown in the lower half of the figure. The first two amino acids of the GS125 mutation correspond to the P box of GR, whereas the third amino acid corresponds to that of TR.

Within the P box, the first two amino acids are most important in defining binding to a DNA-response element. Therefore, based on the differences between the P box of GR and TR, we constructed a P box mutant that substituted the first two amino acids in TR with the first two amino acids of GR (EGG to GSG) (Fig. 1). The GS125 TR-beta mutant had markedly impaired function on both positive and negative TRE due to disrupted DNA binding. However, this unique DBD mutant was fully functional when tested on a composite TR/GR DNA response element. We demonstrate, therefore, that DNA binding by TR-beta is required on all nTREs known to regulate the H-P-T axis.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Plasmid Constructions-- Point mutations in the thyroid hormone receptor beta  were constructed by site-directed mutagenesis in the context of the TR-beta 2 isoform. GS125 mutant TR-beta 2 cDNA was inserted into the pKCR2 (pSG5) expression vector at an EcoRI site; wild type TR-beta 1 and TR-beta 2 were constructed in a similar way and have been described previously (34, 35). The construction of thyroid hormone-responsive luciferase reporter genes (DR+4-TKLuc, LAP-TKLuc, and PAL-TKLuc) has been reported previously (23). Negatively regulated gene promoters in the identical luciferase reporter (pA3Luc) have also been described: human TRH -900 to +55, human TSH-alpha -846 to +44, and human TSH-beta -1192 to +37 bp (36). The hybrid TR/GRE 3, as two copies, was inserted upstream of TK 109 in pA3Luc. The GAL4 reporter plasmid UAS-TK-Luc contains two copies of the GAL4 recognition sequences upstream of TK109 in pA3Luc. Construction of nuclear receptor corepressor (NCoR), steroid receptor coactivator-1 (SRC-1), and RXR-alpha Gal4BD fusion expression vectors was described previously (37). The VP16AD-GS125 mutant TR-beta 2 was created by exchanging appropriate restriction fragments within the VP16AD-TR-beta 2 construct, which has been reported previously (38).

Electrophoretic Mobility Shift Assay-- The oligonucleotide sequences of DR+4 and TRH site 4 probe were reported previously (13, 37). Hybrid TR/GR-response elements, TR/GRE 1, 2, and 3-DR+4 were designed to contain an intact 5' half-site and a mutant 3' half-site separated by a four-nucleotide gap. This strategy was based on polarity studies of the DR+4 element where it has been shown that RXR binds to the 5' and TR binds to the 3' half-sites in a DR+4 (39, 40). Wild type or mutant TRs were transcribed/translated in vitro in reticulocyte lysate (Promega Corp., Madison WI) using T7 polymerase. To quantify protein production, 35S incorporation and direct visualization on SDS-PAGE were used. Four µl of TR-beta and 2 µl of in vitro translated RXR-alpha or an equivalent amount of unprogrammed lysate were used in each reaction with 32P-radiolabeled probe. Binding reactions were performed in 20 µl of binding buffer containing 20% glycerol, 20 mM HEPES, pH 7.6, 50 mM KCl, 1 mM dithiothreitol, 1 µg of poly(dI-dC), and 0.1 µg of salmon sperm DNA at room temperature for 20 min. The protein-DNA complexes were resolved on 5% nondenaturing polyacrylamide gels and visualized after autoradiography.

Western Blot Analysis-- Whole cell extracts were prepared with RIPA Buffer (41) including 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 40 µl/ml Protease Inhibitor Mixture (Roche Molecular Biochemicals). Proteins were resolved by SDS-PAGE and transferred onto nitrocellulose transfer membranes (Protran®; Schleicher & Schuell, Keene, NH). Immunodetection was performed using mouse monoclonal TR-beta antibody (MA1-215; Affinity Bioreagents, Golden, CO) and anti-mouse, horseradish peroxidase-conjugated IgG (Amersham Biosciences). Protein bands were visualized using ECL-Plus kit (Amersham Biosciences) according to the manufacturer's instructions.

Avidin-Biotin Complex DNA Binding (ABCD) Assay-- Whole cell extracts from GH3 cells or transfected 293T cells were incubated with 3 µg of biotinylated double-stranded DNA at 4 °C for 4 h with constant rotation. Sense and antisense TRE oligonucleotides were 5'-biotinylated as follows: rGH sense (5'-ttt cgg tgg aaa ggt aag atc agg gac gtg acc gca gga gag caa a-) and antisense (5'-ttt gct ctc ctg cgg tca cgt ccc tga tct tac ctt tcc acc gaa a-); hTSH-beta sense (5'-ttt ttt ggg tca cca cag cat ctg ctc acc aat gca aag taa gaa a-) and antisense (5'-ttt ctt act ttg cat tgg tga gca aga tgc tgt ggt gac cca aaa aa-); and mutated hTSH-beta sense (5'-ttt ttt ttt tca cca cag cat ctg ctc acc aat gca aag taa gaa a-) and antisense (5'-ttt ctt act ttg cat tgg tga gca aga tgc tgt ggt gaa aaa aaa aa-). The DNA-protein suspensions were precipitated with streptavidin-agarose (Invitrogen) for 1 h, washed with RIPA Buffer, and re-precipitated by centrifugation. The bound materials were eluted and separated with boiling and brief centrifugation and then resolved by SDS-PAGE. Western blot analysis was performed as described previously (41).

ChIP Assays-- ChIP assays were performed essentially as described previously (42) with the following modifications. 293T cells were transfected with 10 µg of hTSH-alpha pA3Luc or TK 109 in pA3Luc and 5 µg of either TR-beta 2 or GS125 mutant TR-beta 2 pKCR2 using the calcium phosphate method. After no treatment or treatment with 10 nM T3, formaldehyde was added at 1% to culture medium, and cells were incubated for 10 min at 37 °C to cross-link proteins to the transfected promoter plasmids. Cells were collected and resuspended in 200 µl of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) with 1 mM phenylmethylsulfonyl fluoride and protease inhibitors. Lysates were cleared by centrifugation and diluted 10-fold with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). 10% of the lysate was kept to quantitate the amount of DNA present in different samples at the PCR analysis as an input. After pre-clearing with a salmon sperm DNA/protein A-agarose/50% slurry (Upstate Biotechnology, Lake Placid, NY), immunoprecipitation with a TR-beta antibody (MA1-215; Affinity Bioreagents) was performed at 4 °C overnight. Immunoprecipitated complexes were collected by salmon sperm DNA/protein A-agarose and followed by sequential washes in low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl immune complex wash buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and TE buffer. Precipitates were eluted with elution buffer (1% SDS, 0.1 M NaHCO3), and 5 M NaCl was added to reverse cross-links at 65 °C for 4 h. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. Pellets were resuspended in TE buffer and subjected to PCR using specific 5' primers for the hTSH-alpha pA3Luc (5'-caa agt gac agc gta ctc tct t) or TK 109 pA3Luc (5'-ccg ccc agc gtc ttg tca tt) and a common 3' luciferase primer (5'-cta gag gat aga atg gcg ccg ggc ctt tct).

Cell Culture and Transient Transfection Assay-- 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with L-glutamine, 10% fetal calf serum, 100 µg/ml penicillin, 0.25 µg/ml streptomycin, and amphotericin. Transient transfections were performed in subconfluent 6-well plates on subconfluent cells; 1.7 µg of reporter construct with 0.1 µg of receptor expression vector or 0.1 µg of pKCR2 vector alone were added to each well using the calcium phosphate technique. 12-15 h after transfection the cells were washed with PBS, and culture medium containing fetal bovine serum treated with anion exchange resin (type AGX-8, analytical grade; Bio-Rad) was added. Twenty four hours after transfection, 10 nM T3 was added, and 42-48 h after transfection culture were harvested and assayed for luciferase activity. Mammalian two-hybrid assays were performed also using the calcium phosphate technique in 6-well plates, with each well receiving 1.7 µg of upstream activating sequence (UAS)-TK luciferase reporter, 0.1 µg of Gal4DBD-SRC-1 NBD, Gal4DBD-NcoR, or Gal4DBD-RXR and 0.1 µg of VP16-TR-beta 1, -TR-beta 2, or VP16-GS125 mutant TR-beta 2.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The DNA Binding Affinity of the GS125 TR-beta Is Reduced on Both Positive TRE and Negative TREs-- Recent cell culture and TR knockout model studies suggest that the beta 2 isoform of the thyroid hormone receptor (TR-beta 2) plays the predominant role in the feedback regulation of the H-P-T axis (43-45). Therefore, we introduced the GS125 mutation into the TR-beta 2 isoform and used it for the following experiments. To assess the DNA binding property of the GS125 mutant, in vitro translated protein was analyzed by gel-shift studies using various radiolabeled TRE probes. A DR+4 probe was employed as a pTRE and a TRH site 4 probe was utilized as a nTRE. The site 4 sequence from human TRH promoter is a well established nTRE (13). As shown in Fig. 2, wild type TR-beta 1 and TR-beta 2 bound to either the DR+4 or TRH site 4 probe as both a homodimer and as a heterodimer with RXR. In contrast the GS125 mutant was unable to bind to either element in the absence or presence of RXR. In data not shown, weak binding of the GS125 mutant to a DR+4 probe was noted if the gel-shift study was performed at 4 °C, but the corresponding wild type TRs also had markedly increasing binding. Thus, the GS125 mutation displayed significantly reduced affinities for both positive and negative TREs.


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Fig. 2.   DNA binding analysis of the GS125 mutant on two different thyroid hormone-response elements. Positive (DR+4 probe) and negative (TRH site 4 probe) TREs were radiolabeled and incubated with in vitro translated TR-beta 1, TR-beta 2, and RXR-alpha . The position of TR homodimeric and heterodimeric complexes are indicated. * nonspecific band in the control lane (Cont.) including unprogrammed reticulocyte lysate.

To demonstrate direct interaction of TR-beta to the negative TRE in vivo, a modified avidin-biotin complex DNA binding (ABCD) assay was employed with non-transfected GH3 cells (Fig. 3). 5'-Biotinylated double-stranded DNA fragments containing either a rat GH pTRE, a human TSH-beta nTRE, or mutated human TSH-beta TRE were incubated with whole cell extracts from GH3 cells in the presence or absence of T3 (21, 47). Bound proteins were precipitated with streptavidin-agarose and tested by Western blot analysis using a specific TR-beta antibody. TR-beta was equally recruited to naturally occurring pTRE and nTRE in the absence or the presence of T3. In the control experiment, a mutation in the human TSH-beta nTRE abrogated TR-beta binding (Fig. 3C). These data clearly demonstrated that DNA binding of TR-beta was DNA-dependent on negative as well as on positive TREs. Next, the DNA-binding affinity of the GS125 TR-beta 2 mutant was evaluated using whole cell extracts from transfected 293T cells. Results in Fig. 3D show that transfected TR-beta 2 bound to the biotinylated positive and negative TRE in a ligand-independent manner. On the other hand, GS125 TR-beta 2 binding to the positive and negative TRE was almost undetectable, which is consistent with the results observed in gel-shift analysis.


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Fig. 3.   DNA binding to TREs in an ABCD assay and modified ChIP assay. A, diagram of a modified avidin-biotin complex DNA binding assay (ABCD assay). B, TRE sequences used in the assay. Positive TRE (rGH; -198 to -158), negative TRE (hTSH-beta ; -3 to +37), and mutated nTRE (mut. hTSH-beta ) were 5'-biotinylated. Arrows indicate the position and orientation of half-sites. ABCD assay was performed using whole cell extracts (WCE) from non-transfected GH3 cells (C) and transfected 293T cells (D). Whole cell extracts were incubated with double-stranded biotinylated pTRE, nTRE, or mutated nTRE in the presence or absence of T3. TR-beta was detected by Western blot analysis using an anti-TR-beta antibody. Whole cell extract was applied to the input lane before performing the binding reaction to demonstrate the location of the TR-beta protein. Negative control (NC) was eluted after the same reaction without adding any biotinylated oligonucleotides. E, ChIP assays of promoters cotransfected with TR-beta 2 or GS125 TR-beta 2 in 293T cells. Arrows indicate the PCR primers used to analyze ChIP samples for human TSH-alpha promoter in the left panel or a TK promoter as a control in the right panel. Cell lysates were immunoprecipitated with TR-beta antibody except negative control (NC) using nonspecific antibody. Input was used to determine total transfected promoter DNA by PCR in the same PCR procedure with ChIP samples.

Because a putative site on the TSH-alpha promoter for TR binding has not been identified, the interaction of TR-beta with the TSH-alpha promoter was investigated using a modified chromatin immunoprecipitation (ChIP) assay in transfected 293T cells (Fig. 3E). In this procedure, DNA is cross-linked, isolated, and subjected to immunoprecipitation with an antibody against a specific protein. By using a TR-beta antibody, association of TR-beta with TSH-alpha promoter was analyzed by PCR with specific primers for the TSH-alpha promoter. For these experiments, the human TSH-alpha promoter pA3Luc plasmid was cotransfected into 293T cells with either TR-beta 2 or GS125 mutant TR-beta 2, and a PCR for the presence of the hTSH-alpha promoter was performed. As demonstrated in the left panel, transfected TR-beta 2 interacted equally well with the TSH-alpha promoter in the absence or presence of T3. As a control experiment, TK 109 pA3Luc plasmid was transfected and immunoprecipitated with the same procedure, and a PCR for the TK promoter was performed. As shown in the right panel, no bands were detected from samples immunoprecipitated with TR-beta antibody. In addition, a nonspecific antibody did not precipitate the hTSH-alpha promoter sequences with transfected wild type TR-beta (NC, Fig. 3E). These results clearly demonstrate that TR-beta 2 associated specifically with the TSH-alpha promoter in cell culture. By contrast, transfected GS125 TR-beta 2 binding to the hTSH-alpha promoter in 293T cells was almost undetectable. Thus, it is clear that the GS125 mutation disrupted the ability of TR-beta to bind to negative thyroid hormone-response elements.

A DBD Mutant of TR-beta 2 Is Defective on Positive and Negative TREs-- Ligand-dependent transcriptional activation by the GS125 mutant TR was examined in transient transfection assays in 293T cells using three different pTREs as reporter constructs. As demonstrated in Fig. 4A, cotransfection of wild type TR-beta 2 increased DR+4 TK-Luc activity by 23-fold after treatment with T3. Interestingly, cotransfection of RXR-alpha significantly reduced T3-mediated activation on this reporter. In contrast, T3 stimulation of this same reporter was not observed with GS125 TR-beta 2 regardless of whether RXR-alpha was cotransfected. Similar results were obtained with the other pTREs: LAP-TKLuc (Fig. 4B) and PAL-TKLuc (Fig. 4C) reporters. The GS125 DBD mutant, therefore, completely abolished T3-dependent transactivation of TR-beta 2 on pTREs.


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Fig. 4.   Function of a TR-beta mutant on pTREs. Ligand-dependent transcriptional activity of GS125 mutant TR-beta 2 on positively regulated reporter gene, DR+4-TK-Luc (A), LAP-TK-Luc (B), and PAL-TK-Luc (C). Equal amounts of RXR-alpha and wild type, mutant TR-beta 2 expression plasmids, or empty vector pKCR2 (0.1 µg) were cotransfected into 293T cells with 1.7 µg of the reporter gene. The data are presented as fold activities, as compared with basal expression (empty vector only, in the absence of T3), and are the means of at least three separate experiments performed in triplicate ± S.E.

To determine whether negative regulation by T3 required TR DNA binding, wild type or GS125 mutant TR-beta 2 plasmids were cotransfected into 293T cells with each of the three negatively regulated gene promoters, TRH-Luc, TSH-beta Luc, and TSH-alpha Luc, and a control promoter, TK109 (Fig. 5). After treatment with T3, wild type TR-beta 2 suppressed these negatively regulated gene promoter activities to ~40% of basal activities, whereas activity from the control reporter was not significantly changed. Moreover, RXR-alpha cotransfection did not significantly increase negative regulation by T3. However, the GS125 mutant TR-beta 2, which lacks DNA-binding activity, paradoxically increased activity by 10-20%. Cotransfection of RXR-alpha did not alter these results. The reduction in transactivation and transcriptional repression was not related to the quantity or stability of the mutant TR-beta 2 protein in tissue culture, as the mutant receptor accumulated to a level similar to the wild type receptor as demonstrated by Western blot analysis of nuclear extracts prepared from transfected 293T cells (Fig. 5A). Thus, the GS125 DBD mutant has no measurable repressive function on negatively regulated genes.


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Fig. 5.   Ligand-induced relative repression on the negative TREs in the H-P-T axis. A, Western blot analysis of nuclear extracts from transfected 293T cells. There are no differences between wild type and mutant TR-beta 2 in their expression level. Wild type, GS125 TR-beta 2, or pKCR2 plasmids were cotransfected into 293T cells with and without equal amounts of RXR-alpha with either TRH, TSH-beta , or TSH-alpha promoter pA3Luc reporter plasmids (C-E, respectively). A similar paradigm was used with TK109 reporter alone as a control (B). Results are expressed as a ratio of activity in the presence versus absence of T3. Data are the mean of at least three separate experiments performed in triplicate ± S.E.

The GS125 DBD Mutant Is Functional on a Composite TR/GR-response Element-- To demonstrate that the GS125 TR-beta 2 is a functional nuclear hormone receptor, we wished to determine whether it had activity on a natural or hybrid thyroid hormone-response element. Because the first two amino acids of the P box in GS125 TR-beta 2 resemble those found in the GR, we tested its function on a naturally occurring glucocorticoid receptor element (GRE), mouse mammary tumor virus promoter in 293T cells. After treatment with T3, the GS125 mutant was unable to activate the mouse mammary tumor virus promoter (data not shown). Next, the GS125 mutant TR-beta 2 was tested for DNA binding on hybrid TR/GR-response elements. Hybrid TR/GREs contained mutations of the third and fourth nucleotides of the downstream half-site hexamer, as shown in Fig. 6B, whereas the upstream TRE half-site and the direct repeat motif with four-nucleotide gap spacing were preserved for binding of heterodimeric partner, RXR. As shown in Fig. 6A, wild type TR-beta 2 and GS125 TR-beta 2 were able to bind to all three hybrid TR/GRE elements as heterodimers with RXR-alpha . For the most part, GS125 TR-beta 2 was able to bind to the hybrid TR/GRE 3 with much higher affinity than that of the wild type TR-beta 2, and this element was chosen for further analysis.


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Fig. 6.   DNA binding and transactivation capacity of wild type and GS125 mutant TR-beta 2 on hybrid TR/GR-response elements. A, gel mobility shift assays were performed with equimolar amounts of wild type and GS125 mutant TR-beta 2 with RXR-alpha using radiolabeled DR+4 derivatives in which the downstream half-site was substituted with the corresponding nucleotide of consensus GRE half-sites as illustrated in B. C, the hybrid TR/GRE 3-DR+4 was inserted upstream of TK109 promoter in pA3Luc as two copies. The resulting reporter construct was cotransfected into 293T cells with wild type, GS125 TR-beta , or pKCR2 with and without RXR-alpha . Data were performed in triplicate and repeated twice. Results are presented as fold activities, as compared with basal expression, and are the mean ± S.E.

A composite TR/GR-response element 3 (AGGTCA cagg AGAACA) was inserted as two copies into the TK-Luc reporter plasmid, and the transcriptional activity of the GS125 TR-beta 2 was tested on this reporter in 293T cells with and without cotransfected RXR-alpha (Fig. 6C). The GS125 mutant in the presence of T3 was very active on this element, whereas wild type TR-beta 2 showed an insignificant increase. Moreover, the transcriptional activation of GS125 TR-beta 2 was significantly increased after cotransfection with RXR-alpha , suggesting that binding of GS125 TR-beta 2 was enhanced in the presence of cotransfected RXR-alpha . These data indicate that the GS125 DBD mutant was expressed in the nucleus and acted as a functional TR on the composite TR/GR-response element.

The GS125 DNA-binding Mutant Can Interact with Transcriptional Cofactors-- To characterize the GS125 mutant function further, interactions between GS125 TR-beta 2 and transcriptional corepressors and coactivators were examined in a mammalian two-hybrid assay. Gal4DBD-NcoR and -SRC-1, which contain their respective nuclear hormone receptor interaction domains, were tested for their ability to interact with wild type and GS125 mutant TR-beta 2 (Fig. 7). Nuclear corepressor protein (NCoR) mediates ligand-independent repression of TRs by forming a complex with histone deacetylase activities in the absence of ligand (48). Gal4-NCoR interacts with VP16-TR-beta 1 and VP16-TR-beta 2 fusion proteins in the absence of T3, resulting in a 5-6-fold activation of the UAS-TK-Luc reporter. In the presence of T3, Gal4-NCoR dissociated from VP16-TRs and as a result the activity of UAS-TK-Luc was reduced to basal levels. Cotransfection of VP16-GS125TR-beta 2 led to the same results as found with both isoform of wild type TR-beta , indicating that the mutant interacted normally with NCoR.


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Fig. 7.   Functional interaction of GS125 DBD mutant with transcriptional cofactors. 293T cells were transiently transfected with 1.7 µg of UAS-TK-luciferase reporter, 0.1 µg of VP16-TR-beta 1, -TR-beta 2, or -GS125 TR-beta 2, and 0.1 µg of GAL4-NcoR (A), GAL4-SRC-1 (B), or GAL4-RXR-alpha (C). The data are presented as fold activities, as compared with basal expression (cotransfected with VP16-blank, in the absence of T3) and are the means of two separate experiments performed in triplicate ± S.E.

In the presence of T3, SRC-1 associates with TRs and mediates ligand-dependent transcriptional activation by TRs. In the presence of T3, a VP-16 fusion of the TR-beta induced activity of UAS-TK-Luc by bound Gal4-SRC-1 by 6-8-fold. Again the GS125 mutant TR-beta 2 displayed an equivalent interaction as compared with wild type TR-beta 2. Finally, the heterodimerization property of GS125 was examined with this assay. Coexpression of Gal4-RXR-alpha and VP16-TR-beta s resulted in a significant increase in reporter gene activity and the addition of T3, enhancing activities by 5-7-fold; equivalent results were observed with the mutant. These data indicate that GS125 DBD mutation did not interfere with corepressor, coactivator, or RXR interactions, indicating that all of the important functional domains of this mutant TR, except DNA binding, are intact.

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ABSTRACT
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In the present study, we characterized the DNA binding domain mutant GS125 TR-beta 2, which has a two amino acid substitution within the P box (GSG for wild type EGG). GS125 TR-beta 2 exhibited functional ligand binding and corepressor and coactivator binding properties similar to wild type TRs, except for its inability to bind to positive and negative TREs. The GS125 DBD mutant was defective in both ligand-induced transactivation on pTRE reporter genes and ligand-induced transrepression on the negatively regulated gene promoters, crucial in regulating the H-P-T axis.

Negative feedback regulation of the H-P-T axis has been well studied because of its physiological importance. A number of reports have identified the element responsible for negative regulation and proposed mechanisms and target cofactors based on the traditional "on-DNA" hypothesis. The negative TRE within the human TSH-beta genes is composed of single half-site motifs present near the TATA box and downstream of the transcription start site, which interact with the TR monomer (21, 22). The human TRH promoter contains three nTREs, including separate half-sites which are responsible for T3-dependent negative regulation and binding of TR. Site 4 in the TRH gene, which is capable of binding not only TR monomers but also TR homodimers and TR-RXR heterodimers, may mediate the enhancement of negative regulation by the RXR (13).

On the contrary, an "off-DNA" hypothesis for negative regulation was proposed by Tagami et al. (49). They suggested that the DNA-binding domain of TR-beta and/or direct DNA binding of TR was not necessary in TR-mediated control of negatively regulated genes. A question was raised, therefore, about whether negative TREs truly existed. These investigators utilized a DBD mutant, however, which would be predicted to disrupt the overall structure of the DBD, making its effect on gene regulation difficult to interpret. In this study, the GS125 DBD mutant provides a strong argument in support of the on-DNA hypothesis even on the TSH-alpha promoter. Clearly, by using this mutant, DNA binding by TR-beta on negatively regulated gene is essential for the T3-mediated transcriptional regulation.

Because nTREs do not contain high affinity TR DNA-binding sites when tested by gel-shift analysis, it has been suggested that these regions might function as a composite site involving the receptor and other transcriptional cofactors. A newer model suggested that T3 induced recruitment of TR and histone deacetylase to the TSH-beta nTRE resulting in negative regulation in GH3 cells (50). This group suggested that TRs interacted directly with HDAC2 through the DNA-binding domain on the negative TRE of the TSH-beta promoter. We employed an avidin-biotin complex DNA binding assay (ABCD assay) using biotinylated oligonucleotides to test the effect of ligand on DNA binding to the same nTRE. We succeeded in detecting direct DNA binding of TR-beta in GH3 nuclear extracts to the nTRE from human TSH-beta gene using Western blot analysis and a TR-beta -specific antibody; T3 had no effect on the extent of TR-beta binding. DNA binding was abolished on a mutant human TSH-beta fragment, indicating that binding was specific to the TSH-beta nTRE (Fig. 3A). These results are in accordance with other studies in which DNA binding is required for TR-beta -mediated negative regulation. We found no evidence to support a T3-mediated recruitment of TR to the TSH-beta nTRE.

A number of studies have reported the effects of P box substitutions on TR DNA binding or the responsiveness of TRs on thyroid hormone-response elements (51-56). However, previously described DBD mutants would be predicted to disrupt the overall alpha -helical structure of the DBD or other functional domain structures. With these DBD mutants, it is difficult to conclude that loss of transcriptional activities is specifically caused by the lack of DNA binding versus other changes in the receptor. In this paper, the GS125 mutation was a P box mutation substituting GSG for the wild type EGG sequence found in TR. This unique DBD mutant TR-beta , which was predicted to bind to a GRE half-site, was able to bind to the hybrid TR/GR-response element and was fully functional on this composite DNA element as a T3-responsive nuclear receptor. T3-dependent transactivation of GS125 TR-beta on the hybrid TR/GR-response element was dramatically increased in the presence of RXR-alpha (Fig. 6). These data indicate that DNA binding of GS125 TR-beta was RXR-dependent in this particular GR/TR composite response element. Finally, the GS125 mutant preserved interactions with transcriptional cofactors as shown in the mammalian two-hybrid assays. These results also support the conclusion that the GS125 mutant TR is a fully functional nuclear hormone receptor.

TRs are encoded by two distinctive genes, c-erbA-alpha and -beta (57, 58). Multiple isoforms of TRs are generated from two genes, and their expressions are regulated in specific temporal and spatial patterns (27). TR-beta 2 has tissue-specific expression in the anterior pituitary gland and the specific areas of the hypothalamus as well as brain and inner ear (7, 46, 59). It has known that TR-beta 2 isoforms play a key role in the feedback regulation of H-P-T axis (43, 44). It is possible that GS125 DBD mutants TR-beta might have various phenotypes in tissue-specific pattern in the in vivo study. For example, it was reported that DNA binding of the GR was necessary for some but not all feedback effects in the hypothalamic-pituitary-adrenal axis (60).

In summary, DNA binding of TR-beta is required for thyroid hormone-mediated transcriptional regulation of both positively and negatively regulated genes. The GS125 DNA-binding mutant establishes the importance of DNA binding in negative regulation of gene transcription mediated by thyroid hormone receptors.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Section of Endocrinology, Dept. of Medicine, the University of Chicago, 5841 S. Maryland Ave., MC 1027, Chicago, IL 60637. Tel.: 773-702-6217; Fax: 773-834-0486.

Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M207264200

    ABBREVIATIONS

The abbreviations used are: H-P-T axis, hypothalamic-pituitary-thyroid axis; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; T3, thyroid hormone; TR, thyroid hormone receptor; TRE, thyroid hormone-response elements; pTRE, positive thyroid hormone response element; nTRE, negatively regulated thyroid hormone response element; RXR, retinoid X receptor; DBD, DNA binding domain; LBD, ligand binding domain; NcoR, nuclear receptor corepressor; SRC-1, steroid receptor coactivator-1; GR, glucocorticoid receptor; ChIP, chromatin immunoprecipitation; UAS, upstream activating sequence; GRE, GR-response element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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