©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand-dependent Antagonism by Retinoid X Receptors of Inhibitory Thyroid Hormone Response Elements (*)

Ohad Cohen , Timothy R. Flynn , Fredric E. Wondisford (§)

From the (1) Thyroid Unit and Charles A. Dana Research Institute and Harvard-Thorndike Laboratory, Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The role of retinoid X receptors (RXRs) on negative thyroid hormone response elements (nTREs) is not well understood. In this report, we demonstrate that an orientation-specific monomeric thyroid hormone receptor (TR) DNA-binding site mediates thyroid hormone inhibition in the thyrotropin subunit gene (TSH-) from human and murine species. Unlike positive TREs, addition of the ligand 9-cis retinoic acid (9-cis RA) to cells transfected with a TR 1 expression vector significantly reduces thyroid hormone inhibition of the TSH- gene, indicating that endogenous retinoid receptors antagonize TR function. Cotransfection of an RXR- but not a retinoic acid receptor- expression vector further antagonizes thyroid hormone inhibition, but only in the presence of 9-cis RA. Antagonism by RXR requires both an intact DNA- and ligand-binding domain. Removal of monomeric TR binding to the TSH- nTRE also requires both RXR domains. A model is proposed whereby monomeric TR is removed from a nTRE by RXR occupied by its ligand 9-cis RA. This is the first report of 9-cis RA-dependent antagonism of thyroid hormone inhibition via negative TREs.


INTRODUCTION

Thyroid hormone mediates its effect through cis-acting response elements. Several investigators (1, 2, 3, 4) have shown that these elements are repeats of a half-site consensus motif ((A/G)GG(T/A)CA). The orientation and spacing of these half-sites determine the specificity of nuclear receptor binding and the outcome of receptor-DNA interactions (4, 5, 6) . Umesono et al.(4) have proposed that a direct repeat of this half-site containing a 4-bp() gap (DR+4) functioned as a positive thyroid hormone response element (pTRE), while DR+3 and DR+5 arrangements functioned as vitamin D and retinoic acid response elements, respectively. Nuclear hormone receptors interact at these sites as either homodimers or heterodimers in association with nuclear auxiliary proteins. Auxiliary proteins have been described for TR (7, 8, 9) . Characterization of these proteins reveal that some (RXRs) are related to retinoic acid receptors (RARs) and bind the ligand 9-cis RA (10, 11, 12, 13, 14) . On positive TREs (pTREs), heterodimer formation between TR and RXR increased T-stimulated transcription (11, 12, 15, 16, 17) . This effect is thought to be mediated by enhanced binding affinity of RXRTR heterodimers to pTREs.

Unlike the role of RXR to increase heterodimer binding and enhance thyroid hormone stimulation from a pTRE, its effect on the negative TRE (nTRE) is not well understood. A major nTRE in the human, mouse, and rat TSH- subunit genes has been described (18, 19, 20, 21) . The human nTRE consists of two consensus half-sites situated 24 bp apart and an additional degenerate half-site (3/6) positioned 4 bp downstream from the first half-site in the first exon (20) . These sites are identical in the mouse and rat TSH- genes, but their individual roles in thyroid hormone inhibition have not been fully characterized. The more 5`-TR half-site (site 1) of this nTRE is of higher affinity than the 3`-half-site (site 2), but both sites appear to be functionally important for thyroid hormone inhibition (20, 21) . Moreover, at site 1, TR structurally interacts with the proto-oncogene c-jun during thyrotropin-releasing hormone stimulation of this gene (22) .

Recently, Naar et al.(5) and Hallenbeck et al. (23) have suggested that negative regulation of the mouse TSH- subunit gene was mediated by formation of unique TR homodimeric or heterodimeric complexes on a somewhat different nTRE. Their proposed nTRE was a DR+2 element located at -18 to -5 bp in the mouse TSH- gene. The more 5`-half-site of this nTRE is relatively degenerate, however, and is not conserved in either the human or rat TSH- subunit genes. We provide evidence that the human and mouse TSH- nTREs bind monomeric TR, and RXR, in a ligand-dependent manner, antagonizes T-mediated inhibition.


EXPERIMENTAL PROCEDURES

Probe Design

Double-stranded P-labeled probes were synthesized using polymerase chain reaction and [-P]dCTP (400 Ci/mM) and incorporated 4-6 [-P]dCTP residues. Unincorporated P was removed by Sephadex G-25 chromatography, and the probes were purified on 5% non-denaturing gels as described (22) . Fig. 1illustrates the DNA probes used in this study.


Figure 1: Wild type and mutant nTREs from the human and mouse TSH- genes utilized in this study. Nucleotide position relative to the start of transcription is indicated above the DNA sequences. TR half-sites in the WT nTREs are boxed, and nucleotide mutations are underlined. Mut, mutant.



Gel Shift Assay

Human c-erbA--1 in pGEM3 and RXR- in pBS (gift of R. Evans, Salk Institute, San Diego) were synthesized in an in vitro transcription/translation reaction (TnT kit, Promega). Unprogrammed translation reactions were used as a control. Quality and quantity of translated proteins were assessed by [S]methionine labeling and SDS-polyacrylamide gel electrophoresis analysis. Binding reactions were performed in a 15-µl volume of binding buffer (20% glycerol, 20 mM HEPES, pH 7.6, 50 mM KCl), 1 mM dithiothreitol, 1 µg of poly(dI-dC), 0.1 µg of salmon sperm DNA. 30,000-50,000 cpm of probe was added to each reaction with 3 µl of programmed or unprogrammed reticulocyte lysate for 30 min at room temperature. ``Supershifts'' were performed using 2 µl of either polyclonal anti-TR-1 (Affinity Bioreagents, Meshanic St., NJ) or anti-c-jun (Santa Cruz Biotechnology, Santa Cruz, CA) with an additional 30 min of incubation at 4 °C. Electrophoresis was performed on a 5% non-denaturing gel containing 5% glycerol as previously described (22) .

Plasmid Construction and DNA Transfections

Mutations noted in Fig. 1were introduced into either a -125/+37-bp human TSH- or a -124/+4-bp mouse TSH- promoter fragment utilizing polymerase chain reaction and mutagenic oligodeoxynucleotides. All constructs were confirmed by restriction enzyme analysis and DNA sequencing. Human or mouse TSH- promoter fragments were cloned into a luciferase vector at the HindIII site as previously described (20) . Human TR 1 and RXR- cDNAs were inserted as EcoRI fragments into the pKCR2 vector as described (22) . RXR- deletion mutant cDNAs were constructed by deleting either an NcoI or StuI fragment from the full-length cDNA and inserting these cDNAs into pKCR2. The human RAR- cDNA in the pCDM8 expression vector was a gift of Dr. Benjamin Neel (Beth Israel Hospital, Boston). Transient transfection of choriocarcinoma cell line JEG-3 was performed using methods previously described (20, 22) . Cell culture medium contained 10% fetal calf serum (Life Technologies, Inc.) treated with either anion exchange resin (50 mg/ml AGX-8, Bio-Rad) for two separate periods of at least 5 h each or anion exchange resin as before with the addition of activated charcoal (100 mg/ml, Sigma) with the second anion exchange resin treatment. The resin and charcoal were then removed, and the serum was filter-sterilized prior to its addition to the cell culture medium.


RESULTS AND DISCUSSION

The Human TSH- nTRE Binds TR Monomer, and RXR- Removes Monomeric TR Binding

The focus of this study was site 1 in the nTRE of the TSH- gene from different species. The WT nTRE and mutations within and around site 1 in the nTRE were tested for their ability to bind TR and RXR in gel mobility studies. The WT nTRE (Fig. 2) bound in vitro translated TR (human c-erbA--1) as a monomer (M); the migration position of a monomeric TR complex was established by comparison to the nTRE containing a DR+4 mutation (Fig. 3), which formed both monomeric and dimeric (D) TR complexes. Using the 3/7 mutation, which eliminated TR binding at site 1, we have previously shown (20, 22) that monomeric TR bound predominantly to this half-site in the WT nTRE. As expected, when site 1 was reversed (M1R), a monomeric TR binding pattern identical to the WT nTRE was observed (data not shown). The nonspecific complex (NS) found with unprogrammed lysate migrated slightly above the heterodimer band and was not ``supershifted'' by the anti-TR-1 antiserum (Fig. 2). Interestingly, the DR+5 DNA fragment (Fig. 4), which contains an additional consensus TR binding half-site 5 bp downstream from site 1 (Fig. 1), bound both one (M) and two monomers (2M) of TR. 10 nM T did not alter monomeric TR binding on the WT nTRE (Fig. 2) but reduced dimeric TR binding on the DR+4 element (Fig. 3), as has been previously reported (24, 25) . Note that unlike the DR+4 probe, 10 nM T did not dissociate the 2 M complex on the DR+5 probe. Polyclonal anti-TR-1 but not anti-c-jun antiserum supershifted the monomeric, two monomeric, and dimeric TR complexes, confirming their identity. In fact, polyclonal anti-TR-1 antibody supershifted two distinct complexes with the DR+4 probe (Fig. 3, arrows), representing a dimeric and monomeric complex but only a single complex with the WT nTRE (Fig. 2, arrow) or the DR+5 probe (Fig. 4, arrow).


Figure 2: Gel mobility shift assay of the wild type nTRE in the human TSH- gene. Radiolabeled DNA probe containing -18 to +37 bp of the human TSH- gene was utilized in a gel mobility shift assay with in vitro translated proteins (3 µl) as noted by the +. 10 nM T, c-erbA-, or c-jun antiserum was used in some binding reactions. UP, unprogrammed; M, monomer; NS, nonspecific band; arrow, supershifted TR complex.




Figure 3: Gel mobility shift assay of the DR+4 mutant nTRE. Radiolabeled DNA probe containing -18 to +37 bp of the human TSH- gene and the DR+4 mutation (Fig. 1) was utilized in a gel mobility shift assay with in vitro translated proteins (3 µl) as noted by the +. 10 nM T, c-erbA-, or c-jun antiserum was used in some binding reactions. M, monomer; D, dimer; HD, heterodimer; arrows, supershifted TR complexes.




Figure 4: Gel mobility shift assay of the DR+5 mutant TRE. Radiolabeled DNA probe containing -18 to +37 bp of the human TSH- gene and the DR+5 mutation (Fig. 1) was utilized in a gel mobility shift assay with in vitro translated proteins (3 µl) as noted by the +. 10 nM T, c-erbA-, or c-jun antiserum was used in some binding reactions. M, monomer; 2M, two monomers; NS, nonspecific band; and arrow, supershifted TR complex.



In vitro translated human RXR- did not form a specific complex with the WT nTRE (Fig. 2) but, when added with TR, eliminated monomeric TR binding. These data suggest that heterodimerization between TR and RXR in solution prevents TR binding to the WT nTRE. Identical results were obtained with the M1R DNA probes (data not shown). In contrast, the DR+4 element (Fig. 3) formed a strong heterodimeric complex when both TR and RXR were added to the binding reaction. The DR+5 element (Fig. 4) behaved like the WT nTRE except that RXR inhibited formation of both monomeric complexes (M and 2M). 10 nM T did not alter heterodimer complexes on the DR+4 probe. Since the ligand for RXR is 9-cis RA (gift of A. Levin, Hoffman-LaRoche, Nutley, NJ), we also tested whether this ligand (13, 14) would alter the pattern of TR or TRRXR heterodimeric binding on these elements. 1 µM 9-cis RA did not alter RXR antagonism of TR monomeric binding on the WT nTRE or DR+5 probes or TRRXR heterodimeric binding on the DR+4 element (data not shown).

The Orientation and Surrounding Nucleotides of Site 1 in the Human TSH- nTRE Are Important for Thyroid Hormone Inhibition

JEG-3 cells were chosen for these studies because they are functionally deficient in TR and have been used to investigate negative T regulation of the common glycoprotein subunit and TSH- subunit genes (22, 26) . Luciferase reporter constructs containing -125 to +37 bp of either the WT or mutant human TSH- nTRE were utilized in these studies. As a positive control, a luciferase reporter construct containing two copies of a T palindromic element (TRETK, Ref. 27, gift of J. L. Jameson, Northwestern University Medical School, Chicago) upstream of a minimal thymidine kinase promoter was utilized. Transfection efficiency was monitored using a cotransfected human growth hormone reporter plasmid (pTKGH); luciferase activity was expressed relative to the basal activity of the human TSH- WT construct, except for the pTRETKLUC activity, which was expressed relative to its own basal activity. Relative luciferase activity (relative light units measured over 20 s/human growth hormone measured in culture medium in ng/ml) of TRETK, WT human TSH-, and ``promoterless'' luciferase constructs was 36, 427, 2115, and 25, respectively.

Transient transfection studies were performed in JEG-3 cells without and with cotransfected human c-erbA--1 (pKCR2-c-erbA-, TR). Luciferase activity from the WT -125/+37-bp human TSH- reporter plasmid (Fig. 5) was increased by cotransfected TR; the addition of 10 nM T reduced luciferase activity by 70%. 10 nM T, in the presence of cotransfected vector, did not inhibit luciferase expression from this construct, confirming the functional absence of TR in this cell line. We have previously shown that mutation of site 1 (Fig. 1, 3/7) abolished T-mediated inhibition of the -125/+37-bp human TSH- reporter plasmid in this cell line in the presence of cotransfected TR (22) . As expected, the TRETK construct was stimulated 16-fold by T in the presence of cotransfected TR (Fig. 5). Note that TR cotransfection resulted in ligand-independent repression, not stimulation, of this construct containing a pTRE.


Figure 5: Functional studies of the WT and mutant nTREs in a transiently transfected human placental cell line (JEG-3). The WT and mutant TREs in the context of -125 to +37 bp of human TSH--luciferase construct were tested in JEG-3 cells. The construct is named for the mutation in the nTRE and corresponds to the nomenclature in Fig. 1. A positive control, the pTRETK plasmid (2 Pal), which contains two palindromic pTREs upstream of a minimal thymidine kinase promoter, was also utilized. Cotransfected plasmid(s) are indicated to the left of each graph. All luciferase activity was corrected for transfection efficiency by measuring secreted human growth hormone in the medium and expressed as % activity of the WT -125 to +37 construct in the absence of cotransfected TR, RXR, and T, except for TRETK, which was expressed relative to its own basal activity. Data are mean ± S.E. of three to six independent determinations.



To determine whether a DR+4 configuration at site 1 would alter T-mediated inhibition, this mutation was constructed in the context of the human TSH- nTRE (Fig. 1). Unlike the WT construct, cotransfection of TR with this construct resulted in a 2-fold reduction in basal activity; T stimulated expression from this construct 5-fold (DR+4, Fig. 5 ). These data, together with those from the WT construct, indicated that unliganded monomeric TR stimulated, while dimeric or heterodimeric TR inhibited, transcription from the WT and mutant TSH- TREs, respectively. Moreover, the WT nTRE was converted to a pTRE by adding a second half-site 4 bp downstream from site 1, indicating that the composition of the TRE and not its location determined the direction of thyroid hormone action.

DR+5 configuration at site 1, however, resulted in a different pattern of expression (DR+5, Fig. 5). Ligand-independent activation by TR was impaired relative to the WT construct, but T-mediated inhibition was preserved. Since the DR+5 probe bound TR as either one or two monomers (see Fig. 4 ), these data indicated that the ability of TR to form homo- or heterodimers (DR+4 arrangement) and not just a second nearby half-site (DR+4 or DR+5 arrangement) was essential for thyroid hormone stimulation. The DR+5 arrangement, unlike the DR+4 element, would bind both RARs and RXRs present in the JEG-3 cell line, and this could explain the difference in ligand-independent activation.

To determine whether the orientation of site 1 is important for T-mediated inhibition, we constructed the M1R mutant (Fig. 1) in the -125/+37-bp human TSH- reporter plasmid. Interestingly, this mutation abolished T-mediated inhibition, even though the only difference between this construct and the WT construct is the orientation of the TR-binding site 1 (M1R, Fig. 1 and Fig. 5). Since this mutant only bound monomeric TR in gel mobility shift experiments, it suggested that the presentation of certain domains of the TR to the transcription complex at site 1 was crucial for T-mediated inhibition.

The Mouse TSH- nTRE Bound TR Monomer and TR/RXR Heterodimers

Unlike the rat and human TSH- subunit genes, Naar et al.(5) have proposed a somewhat different nTRE in the mouse. Their proposed mouse TSH- nTRE includes site 1 present in the rat and human genes but also includes a second degenerate half-site more 5` to site 1, which is not found in either the rat or human genes (Fig. 1). When tested in the gel mobility shift assay, the WT mouse TSH- DNA fragment (identical to that in Ref. 5) formed a specific complex with TR that migrated at the position of a TR homodimer (Fig. 6, lane1). Using the WT mouse probe, unprogrammed lysate (data not shown) and the RXR only (lane7) did not form this band, confirming it was due to TR binding. Two lines of evidence suggested, however, that this was not a true homodimer but represented binding of two monomers to this element. First, 10 nM T dissociated TR homodimers on the mouse DR+4 element and resulted in strong monomeric binding (compare Fig. 6, lanes5 and 6) but did not dissociate the WT mouse nTRE or an idealized mouse DR+2 element (Fig. 6, lanes1 and 2 and lanes3 and 4, respectively). A lack of TR-TR interaction on the WT and DR+2 elements can be inferred because T dissociated TR homodimers on the DR+4 but not the WT or DR+2 elements. Furthermore, T altered homodimeric but not monomeric TR binding on the DR+4 element, indicating monomeric TR-DNA interactions were not disrupted by T. Second, we were unable to cross-link TR on the mouse nTRE or an idealized DR+2 element using 1,6-bismaleimidohexane and previously described methods (data not shown and Ref. 20). These data are consistent with that of Naar et al.(5) who demonstrated no significant cross-linking of TR on the WT mouse TSH- nTRE.


Figure 6: Gel mobility shift assay of the WT and mutant mouse TSH- nTREs. Radiolabeled DNA probes, containing -20 to +4 bp of either the WT or mutant (DR+2 and DR+4) mouse TSH- gene (Fig. 1), were utilized in gel mobility shift assay with in vitro translated proteins as noted by the +. The position of the two monomer (2M), homodimer (D), or heterodimer (HD) complex is indicated. NS, nonspecific band.



Mutation of the 5`-Degenerative Half-site in the Mouse TSH- nTRE Does Not Alter Thyroid Hormone Inhibition

Luciferase reporter constructs containing -125 to +37 bp of either the human TSH-, mouse TSH-, or a mutant mouse TSH- gene nTRE were utilized in these studies. As shown in Fig. 7, the WT human construct was inhibited 90% by 10 nM T in the presence of cotransfected TR in these experiments. The WT mouse construct was inhibited 77% by 10 nM T in the presence of cotransfected TR (Fig. 7). The more 5`-degenerative half-site in the mouse nTRE, however, was not important in mediating thyroid hormone inhibition, since the mutant mouse nTRE (shown in Fig. 1 ), which resembles the human nTRE, was inhibited to a greater extent (93%) than the WT mouse construct by 10 nM T (Fig. 7). These data indicate that the core monomeric site in the mouse TSH- gene nTRE and not the DR+2 arrangement dictates negative regulation.


Figure 7: Functional comparison of the WT human, mouse, and mutant mouse (MT) TSH- promoters in JEG-3 cells. The WT human TSH- nTRE in the context of the human promoter (-125 to +37 bp) or the WT and mutant mouse TSH- nTREs in the context of -124 to +4-bp promoter were tested in JEG-3 cells. The construct named corresponds to the nomenclature in Fig. 1. Data are expressed as a percent of a relative luciferase activity ratio (+T/-T). Data are mean ± S.E. of three to six independent determinations.



RXR Does Not Antagonize Thyroid Hormone Inhibition in the Absence of 9-cis RA

Having defined the importance of the core monomeric TR-binding site (site 1) of the mouse and human TSH- gene nTREs, we next explored the role of RXR in thyroid hormone inhibition. Both Hallenbeck et al.(23) and Carr and Wong (28) have reported that cotransfection of RXR- and TR expression vectors abolished thyroid hormone inhibition of either the mouse or rat TSH- gene nTRE. It is unclear, however, how the cell culture medium was treated in these studies to remove endogenous thyroid hormones and retinoids present in the fetal calf serum. We initially performed experiments with the WT human promoter in medium containing fetal calf serum that was treated with an anion exchange resin to remove endogenous thyroid hormones. When both TR and RXR expression vectors were cotransfected in JEG-3 cells, no significant thyroid hormone inhibition of the WT human construct was observed (2% inhibition by 10 nM T).

To investigate further the effect of RXR on thyroid hormone receptor function, experiments were performed with medium containing serum treated with both anion exchange resin and activated charcoal to remove both endogenous thyroid hormones and retinoids. We first compared the effect of RXR- or RAR- and their ligand 9-cis RA on TR activation of the WT human construct (Fig. 8). Ligand-independent activation by TR was significantly reduced by RXR cotransfection but only in the presence of 9-cis RA. In contrast, RAR cotransfection actually enhanced ligand-independent activation by TR in the absence of 9-cis RA and had no significant effect in the presence of 9-cis RA. These data indicate that ligand-bound RXR functionally removes the ligand-independent activation function of TR.


Figure 8: Effect of 9-cis RA on ligand-independent activation by TR of the WT human TSH- promoter after cotransfection of RXR and RAR expression vectors. The effect of 9-cis RA on basal expression of the WT human TSH- nTRE in the context of the human promoter (-125 to +37 bp) was tested in JEG-3 cells after cotransfection of either TR, TR/RXR, or TR/RAR expression plasmids. Data are expressed as a percent of relative luciferase activity in the absence of 9-cis RA after cotransfection of TR alone. Data are mean ± S.E. of six independent determinations.



We next compared the ability of RXR- or RAR- to antagonize thyroid hormone inhibition of the WT human construct in the absence or presence of 9-cis RA (Fig. 9). As expected, transfection of either an RXR- or RAR- expression vector alone resulted in no T inhibition in either the absence of presence of 9-cis RA. Transfection of TR alone resulted in 75% T inhibition, which was reduced to 50% T inhibition after the addition of 9-cis RA. JEG-3 cells express the RXR- isoform almost exclusively (29) ; these data indicate that endogenous RXR- in JEG-3 cells can antagonize thyroid hormone inhibition when occupied by its ligand, 9-cis RA. This effect was specific for RXR since cotransfection of RXR and TR completely abolished T inhibition in the presence of 9-cis RA, while cotransfection of RAR and TR yielded results similar to TR transfection alone.


Figure 9: Effect of 9-cis RA on T-mediated inhibition of the WT human TSH- promoter after cotransfection of RXR and RAR expression vectors in JEG-3 cells. The effect of 9-cis RA on T-mediated inhibition of the WT human TSH- nTRE in the context of the human promoter (-125 to +37 bp) was tested in JEG-3 cells after cotransfection of either TR, RXR, TR/RXR, RAR, or TR/RAR expression plasmids. Data are expressed as a percent of a relative luciferase activity ratio (+T/-T). Data are mean ± S.E. of three to six independent determinations.



These data are in contrast to those obtained with the TRETK construct. After cotransfection of RXR and TR, T stimulation of this construct was similar in the absence or presence of 9-cis RA (9.3 ± 0.4- versus 9.5 ± 7-fold, respectively). Rosen et al.(30) demonstrated that cotransfection of RXR enhanced T stimulation on a palindromic element, and this enhancement was dependent on 9-cis RA. While our data do not confirm 9-cis RA enhancement of T stimulation in JEG-3 cells, they do demonstrate an antagonistic role for the same ligand in T inhibition in these cells.

RXR Antagonism of Thyroid Hormone Inhibition Requires an Intact DNA- and Ligand-binding Domain

To determine the mechanism of RXR antagonism of TR action, two deletions of the RXR- isoform were constructed and translated in a coupled in vitro transcription/translation reaction. As shown in Fig. 10 , these mutants were synthesized to a similar degree in the lysate as determined by [S]methionine labeling. The DBD mutant lacks amino acids 29-197, and the LBD mutant lacks amino acids 403-462 and are correspondingly smaller in molecular size than WT RXR- of approximately 52 kDa. Both mutants contain the region important in nuclear localization in this superfamily or receptors (31) , and these mutants localize to the nucleus based on deletion studies with the RXR- isoform (32) .


Figure 10: RXR mutants used in this study. An autoradiogram of S-labeled full-length human RXR- and its deletion mutants is shown. The location of the deletion is illustrated. RXR-LBD (StuI fragment removed) deletes part of the ninth heptad in the C-terminal region, and RXR-DBD (NcoI fragment removed) deletes part of the N terminus and the entire DNA-binding domain.



We next determined whether these isoforms would antagonize thyroid hormone inhibition by TR of the WT human TSH- promoter. JEG-3 cells were cotransfected with TR and RXR- expression vectors as noted in Fig. 11. As expected, cotransfection of the full-length RXR- expression vector abolished T inhibition in the presence but not in the absence of 9-cis RA. In contrast, both mutant RXRs were defective in this property. Cotransfection of the LBD mutant resulted in no significant additional antagonism of thyroid hormone inhibition by 9-cis RA compared with cotransfection of TR alone. This result was expected since the LBD mutant lacks the ninth heptad repeat in the C terminus and does not bind as a heterodimer with TR (33, 34) . Cotransfection of the DBD mutant resulted in some additional antagonism of T inhibition in the presence of 9-cis RA as compared with TR alone but clearly less than the full-length RXR.


Figure 11: Effect of 9-cis RA on T-mediated inhibition of the WT human TSH- promoter after cotransfection of RXR mutant expression vectors in JEG-3 cells. The effect of 9-cis RA on T-mediated inhibition of the WT human TSH- nTRE in the context of the human promoter (-125 to +37 bp) was tested in JEG-3 cells after cotransfection of either TR, TR/RXR, TR/RXR-DBD, or TR/RXR-LBD expression plasmids. Data are expressed as a percent of a relative luciferase activity ratio (+T/-T). Data are mean ± S.E. of three to six independent determinations.



Given that both an intact DNA and ligand binding domain of RXR were necessary for ligand-dependent antagonism of thyroid hormone inhibition, we wanted to determine how these RXR mutants affected heterodimer binding to both positive and negative TREs. Both RXR mutants were defective in heterodimer formation with TR on a DR+4 probe (Fig. 12) but did not affect homodimer binding to this element. In contrast, the WT RXR and DBD mutant (to a lesser extent) but not the LBD mutant removed monomeric TR binding to the WT TSH- nTRE (Fig. 12). Addition of 9-cis RA did not alter the effect of RXR- and its mutants on either the DR+4 or TSH- probes (data not shown). Because reticulocyte lysate should contain 9-cis RA, it is unclear whether 9-cis RA affects heterodimer formation between RXR and TR on DNA or in solution.


Figure 12: Gel mobility shift assay of RXR mutants on a DR+4 and WT human TSH- nTRE. Radiolabeled DNA probes, containing either the DR+4 mutant or WT human TSH- gene (Fig. 1), were utilized in gel mobility shift assay with in vitro translated proteins (3 µl) as noted by the +. The position of the a monomer (M), homodimer (D), or heterodimer (HD) complex is indicated. NS, nonspecific band.



A Model of RXR Antagonism of Thyroid Hormone Inhibition

Fig. 13 is a model of RXR action on positive and negative TREs. RXR enhances TR binding to pTREs via a DNA-binding element containing appropriately spaced half-sites (A) in gel shift studies. Both T and 9-cis RA may activate transcription by altering the conformation of the heterodimer and allow it to interact with either an adaptor protein or the basal transcriptional machinery (B). In contrast, we suggest that the TSH- nTRE is a monomeric TR DNA-binding element in human and murine species, binding one or several molecules of TR. RXR removes TR binding to this nTRE and antagonizes both ligand-independent activation by TR as well as T-mediated inhibition (C and D). The importance of the monomer in this process is further strengthened by the orientation dependence of the half-site in the nTRE for inhibition and the ability of a second appropriately spaced half-site to convert a nTRE to a pTRE. RXR antagonism of negative TREs is strictly dependent on an intact DNA- and ligand-binding domain of RXR and the ligand 9-cis RA. In conclusion, this is the first demonstration of 9-cis RA-dependent antagonism of thyroid hormone inhibition via negative TREs.


Figure 13: Model of RXR action on positive and negative TREs. TR and its ligand T are shown as ovals, and RXR and its ligand 9-cis RA are shown as rectangles.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK-43653. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Thyroid Unit, RN-330, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2920; Fax: 617-667-2927.

The abbreviations used are: bp, base pair(s); RXR, retinoid X receptor; 9-cis RA, 9-cis retinoic acid; nTRE, negative thyroid response element; pTRE, positive thyroid response element; TR, thyroid hormone receptor; WT, wild type; TSH-, thyrotropin subunit; DR, direct repeat; RAR, retinoic acid receptors.


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