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Endocrinology Vol. 139, No. 2 640-650
Copyright © 1998 by The Endocrine Society

ARTICLES

Nuclear Corepressors Enhance the Dominant Negative Activity of Mutant Receptors That Cause Resistance to Thyroid Hormone1

Tetsuya Tagami and J. Larry Jameson

Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Tarry 15–709, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: ljameson{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The syndrome of resistance to thyroid hormone (RTH) is caused by multiple distinct mutations in the ligand-binding domain of the thyroid hormone receptor-ß (TRß). Although the mutant receptors are transcriptionally inactive, they inhibit normal receptor function in a dominant negative manner to cause hormone resistance. Recently, a group of transcriptional cofactors, referred to as corepressors (CoRs), was shown to induce ligand-independent silencing of genes that contain positive T3 response elements. CoRs also play a role in the ligand-independent basal activation of genes that are negatively regulated in response to T3. We hypothesized that CoR might play a role in the dominant negative inhibition by TRß mutants that cause RTH. In gel mobility shift assays, RTH mutants retained interactions with CoRs even in the presence of T3, whereas the ligand dissociated CoR from wild-type TRß. Using Gal4-TR chimeric receptors and a VP16-CoR fusion protein in an interaction assay, a strong positive correlation was found between mutant receptor interactions with CoR and transcriptional silencing activity. A mutation (P214R) that impairs CoR interactions with TR was introduced into the RTH mutants to assess the role of CoR in dominant negative activity. In transient transfection assays, introduction of the P214R CoR mutation decreased RTH mutant silencing of positively regulated genes and basal activation of negatively regulated genes. The dominant negative activity of several different RTH mutants, studied by cotransfection with wild-type receptor, was greatly diminished by the CoR mutation, and this effect was seen with both positively and negatively regulated genes. These results suggest that CoR interactions play a critical role in the dominant negative effect of RTH mutants and support the idea that these proteins are involved in the regulation of genes that are positively as well as negatively regulated by T3.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID hormone receptors (TRs) function as ligand-regulated transcription factors that increase or decrease the expression of target genes (1, 2). In the unliganded state, TRs suppress or silence the basal activity of promoters that contain positively regulated hormone response elements (3, 4, 5). The addition of ligand (T3) reverses silencing and stimulates transcription to a level that is even greater than the original basal state. In contrast, the basal activity of negatively regulated genes is stimulated by unliganded receptor, and transcription is repressed after the addition of T3 (6, 7, 8). Recently, nuclear corepressors (CoRs), variously termed nuclear receptor corepressor (NCoR) (9, 10), silencing mediator for retinoid and thyroid hormone receptors (SMRT) (11), thyroid receptor-associating cofactors (12), and thyroid receptor-interacting proteins (13), have been identified. The CoRs interact with the ligand-binding domain (LBD) of nuclear receptors and mediate ligand-independent repression. Several mutations in the so-called CoR box at the amino-terminal end of the LBD disrupt binding to CoRs (9, 10, 11), and there is evidence for additional interactions with more carboxyl-terminal regions of the LBD (14, 15). In a previous study, a mutation (P214R) that corresponds to the CoR box (amino acids 211–240) in the hinge region of human TRß was shown to cause the loss of basal activation of negatively regulated genes as well as silencing of positively regulated genes (16). This finding suggests that CoRs also play a role in basal activation of genes that are negatively regulated in response to T3.

Resistance to thyroid hormone (RTH) is an autosomal dominant disorder that is caused by mutations in the TRß gene (17, 18). Most TRß mutations reduce binding to T3, although some appear to impair transcriptional activity despite near-normal T3 binding (19, 20, 21). Consistent with the dominant mode of transmission, the mutant receptors interfere with the function of normal TRs by a dominant negative mechanism (22, 23, 24). Although the mechanism of dominant negative activity is still being investigated, most data support the idea that mutant receptors retain the ability to bind to DNA and block access of normal TRs to their target genes (25).

Although hormone resistance occurs to varying degrees in all tissues, the diagnosis of RTH is based primarily upon abnormalities in the TRH-TSH-T3 axis (26). Specifically, RTH is characterized by elevated levels of free thyroid hormone without evidence of appropriate suppression of TSH. The degree of hypothalamic-pituitary resistance establishes a set-point that defines the circulating hormone levels that act on all other tissues. For these reasons, it is of great interest to examine negatively regulated as well as positively regulated genes when considering the targets for the dominant negative effects of mutant receptors.

Because CoRs play an important role in the function of unliganded TRs and are dissociated upon the addition of T3, we hypothesized that interactions with CoRs might be important for the dominant negative activity of RTH mutants. In this report, we analyzed the effects of a CoR mutant of TRß (P214R) on the dominant negative effect of RTH mutants using positively and negatively regulated T3-responsive promoters.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction and receptor mutagenesis
The plasmid PAL-TK-Luc contains two copies of a palindromic thyroid hormone response element (TRE; 5'-gatctcAGGTCATGACCTgagatc-3') upstream of the thymidine kinase promoter (TK109) in the pA3 luciferase (Luc) vector (27). DR4-SV40-Luc contains four copies of a direct repeat TRE (5'-agcttcAGGTCActtcAGGTCActcga-3') upstream of simian virus 40 (SV40) promoter in the pGL3 Luc vector (Promega, Madison, WI). TSH{alpha}-Luc contains 846 bp of the 5'-flanking sequence and 44 bp of exon I from the human glycoprotein hormone {alpha}-subunit gene in pA3-Luc (28). TSHß-Luc contains 128 bp of the 5'-flanking sequence and 37 bp of exon I from the human TSH ß-subunit gene in pA3-Luc (16). The pCMV-TEF expression vector (thyrotroph embryonic factor) (29) was provided by M. G. Rosenfeld (University of California, San Diego, CA). The Gal4 reporter plasmid, UAS-TK-Luc, contains two copies of the Gal4 recognition sequence (UAS) upstream of TK109 in pA3-Luc.

The mutant human TRß complementary DNAs (cDNAs) were prepared by oligonucleotide-directed mutagenesis and verified by DNA sequencing as described previously (28) (Fig. 1Go). The numbering of the amino acid residues of TRß is based on a consensus nomenclature (30). Mutant and wild-type receptor cDNAs were expressed using a Rous sarcoma virus (RSV)-driven expression vector (31). The double mutations with P214R were made by inserting the PstI-PflMI fragment of the P214R mutant into the RTH mutant receptor cDNAs. An artificial EcoRI site was introduced into the TRß cDNA to allow insertion of an EcoRI fragment encompassing the LBD of TRß (residues 174–461) in-frame with the Gal4 DNA-binding domain (DBD) in pSG424 (32). The Gal4-P214R and other TR mutants were created by exchanging appropriate restriction fragments into the Gal4-TRß construct. The pCMX-NCoR expression vector was provided by M. G. Rosenfeld (University of California, San Diego, CA) (9). An artificial EcoRI site was introduced into the NCoR cDNA to allow insertion of an EcoRI fragment (residues 1552–2453) including the interaction domain of NCoR in-frame with the VP16 activation domain in pAASV (32). The pCMX-NCoR-ID (internal ATG at amino acid 1579) expression vector was created by deleting a NotI-BstXI fragment from pCMX-NCoR.



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  		Figure 1. Structures of TR mutants. The full length wild-type TRß is depicted at the top of the figure. The central DBD is shaded, and the carboxyl-terminal LBD is indicated. In other constructs, the LBD of TRß was fused to the DBD of Gal4. Amino acid substitutions are denoted by black dots, and a nine-amino acid carboxyl-terminal deletion mutant (P453X) is indicated by {Delta}9. The carboxyl-terminal half of the NCoR protein containing two interaction domains (ID) was also fused to the activation domain of VP16 to create VP16-NCoR.

 
Electrophoretic mobility shift assay
The DNA-binding and NCoR interaction properties of in vitro translated TR and mutant receptors were studied using a radiolabeled TRE, as described previously (33). The sequences of the TRE oligonucleotides were: DR4-TRE sense, 5'-agcttcAGGTCActtcAGGTCAc-3'; and anti-sense, 5'-agTCCAGTgaagTCCAGTgagct-3'. In vitro transcribed/translated receptors (3 µl) were preincubated with in vitro translated human retinoid X receptor-{alpha} (RXR{alpha}) (3 µl) and/or NCoR-ID (4 µl) or with unprogrammed lysate. Reactions were incubated in 24 µl binding buffer [20 mM HEPES (pH 7.8), 50 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM dithiothreitol (DTT), and 40 µg/ml poly(dI-dC)] in the presence or absence of 20 nM T3 at room temperature for 15 min. 32P-Labeled TRE oligonucleotides were added to the reaction and incubated at room temperature for an additional 30 min. The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel using 0.5 x TBE (45 mM Tris-borate and 1 mM EDTA) buffer containing 2.5% glycerol.

Whole cell lysates from transfected TSA-201 cells were prepared by three cycles of freeze-thaw lysis in 20 mM Tris-HCl (pH 7.5), 0.5 M KCl, 2 mM DTT, 20% glycerol, and 1 mM phenylmethylsulfonylfluoride. Cell extracts were prepared by centrifugation at 10,000 x g for 30 min at 4 C, and supernatants were stored at -20 C. Cell extracts (5 µg) were preincubated with in vitro translated human RXR{alpha} (3 µl) or with unprogrammed lysate in 24 µl of a modified binding buffer [20 mM HEPES (pH 7.8), 100 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM DTT, and 100 µg/ml poly(dI-dC)] at 4 C for 15 min.

Tissue culture and transient expression assays
TSA-201 cells, a clone of human embryonic kidney 293 cells (34), were grown in Optimem (Life Technologies, Grand Island, NY) supplemented with 4% Dowex resin-stripped FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were plated in 12-well dishes 16 h before transfection and were transfected by the calcium phosphate method (35). Transfection reactions contained 10–250 ng reporter plasmids together with 10–300 ng of the receptor expression plasmids. When TSHß-Luc was transfected, 5 ng of the TEF expression vector were added (29). The total amount of each receptor construct was maintained constant in each reaction by the addition of the control plasmid without receptor. After exposure to the calcium phosphate-DNA precipitate for 8 h, Optimem with 4% resin-stripped FBS was added, with or without 1 nM T3. Cells were harvested after 40 h for measurement of luciferase activity (36). Transient assays were performed at least in duplicate transfections, and the results are expressed as the mean ± SD from at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Structures of receptor mutants
The structures of the mutant TRß used in this study are depicted in Fig. 1Go. The P453X mutant contains a nine-amino acid deletion at the carboxyl-terminus that deletes a critical part of a trans-activation domain (AF-2) (37) and eliminates T3 binding (28). A Pro to His substitution at amino acid 453 (P453H) (18) and a Gly to Arg substitution at amino acid 345 (G345R) (17) were two of the original mutations described in RTH. The G345R mutant does not bind T3, whereas the P453H mutant binds T3 with about 10% normal affinity (23). Both RTH mutants have been shown to act in a dominant negative manner to inhibit T3 regulation of target genes (19, 22, 23). An artificial mutation (P214R) was created in human TRß that is analogous to a revertant mutation in v-erbA that fails to silence TR-regulated genes (38). This substitution corresponds to the P160R mutation in rat TR{alpha}1 that has been shown to disrupt interactions with the CoR, SMRT (11). This region also corresponds to the so-called CoR box (amino acids 211–240) in the hinge region of human TRß that has been shown to interact with another CoR, NCoR (9).

CoR-binding properties of TRß mutants
The interaction of CoRs with wild-type and mutant receptors was examined using gel mobility shift assays and an interaction assay that is a variation of the two-hybrid assay performed in mammalian cells.

A direct repeat (DR4) of the TRE was used for the gel mobility shift assays. The ability of the carboxyl-terminal half of the NCoR protein (NCoR-ID), which contains two interaction domains with nuclear receptors (39), was used to supershift TR complexes (40). Initially, studies were performed with TR{alpha} and TRß in the absence or presence of RXR{alpha} to generate a series of distinct monomer, homodimer, and RXR-TR heterodimer complexes (Fig. 2AGo). A control using NCoR-ID in the absence of TRs showed no binding to the DR4 element (Fig. 2AGo, lane 1). In the absence of RXR, TR{alpha} formed monomer and homodimer complexes with DR4 (Fig. 2AGo, lane 2), whereas TRß formed predominantly homodimers and few monomer complexes (lane 6). The addition of RXR results in a slower mobility RXR-TR heterodimer complex with both receptor isoforms (TR{alpha} and TRß), causing a reduction in the amount of homodimers (Fig. 2AGo, lanes 3 and 7). In the absence of RXR, the addition of NCoR-ID supershifts the TR bands, resulting in very low mobility complexes (lanes 4 and 8). Consistent with previous studies (40), this finding suggests that TR alone can interact with the NCoR-ID and does not require the presence of RXR. In the presence of RXR, NCoR-ID causes a supershift that primarily decreases the amount of TR homodimer, with less effect on the RXR-TR heterodimer complex (Fig. 2AGo, lanes 5 and 9). The decrease in the amount of homodimers caused by the addition of RXR may account for the reduced amount of complex that is supershifted by NCoR-ID.



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  		Figure 2. Interaction of NCoR with wild-type and mutant thyroid hormone receptors in gel mobility shift assays. NCoR-ID binding to in vitro translated TR{alpha} or TRß wild-type (A) or mutant receptors (B) was analyzed with or without in vitro translated RXR{alpha}. The DNA binding site is 32P-labeled DR4-TRE, and the concentration of T3 is 20 nM. The positions of TR-TR homodimer, the TR heterodimer with RXR, and the complexes supershifted by NCoR-ID are indicated. URL, Unprogrammed reticulocyte lysate.

 
Having characterized the TR complexes supershifted by NCoR-ID, interactions were examined between RTH mutants and NCoR-ID. The TRß homodimer complex was dissociated by the addition of 20 nM T3 and also prevented the formation of the TRß complex with NCoR-ID (Fig. 2BGo, lane 4). The addition of T3 did not dissociate the heterodimer complex, and no interaction of the heterodimer was observed with NCoR-ID (Fig. 2BGo, lane 5). The RTH mutant, G345R, formed a complex with NCoR-ID independent of the presence of T3 (Fig. 2BGo, lanes 6–9). The P214R CoR mutant (Fig. 2BGo, lanes 10 and 11) and its double mutant with G345R (lanes 14 and 15) revealed reduced interaction with NCoR-ID compared with wild-type TR or the G345R RTH mutant. Similar results were obtained using P453X or P453H as double mutants with P214R (data not shown).

Interactions between various TR mutants with CoRs were also examined using a mammalian two-hybrid assay. The LBD (residues 174–461) of TRß was fused to the DBD of the yeast transcription factor, Gal4. The carboxyl-terminal half of the NCoR protein, which contains two TR interaction domains, was fused to the transcriptional activation domain of VP16. The reporter gene, UAS-TK-Luc, contains two Gal4-binding sites and was used to assess in vivo interactions between Gal4-TR and VP16-NCoR-ID in TSA-201 cells (Fig. 3AGo). Gal4-TRß was used initially to test the interaction assay. Relative to the Gal4 DBD alone, Gal4TRß was stimulated 44-fold by the addition of VP16-NCoR. In contrast, the P214R CoR mutant was stimulated 12-fold by VP16-NCoR, suggesting that this single point mutation abrogates, but does not completely eliminate, TR interactions with NCoR (16). VP16-NCoR interactions were also examined with each of the RTH mutants and the double mutants with P214R. Strong NCoR interactions were detected for P453X and P453H (65- and 70-fold, respectively), whereas the interaction with G345R was less pronounced (21-fold). Introduction of the P214R mutation into each of the RTH fusion proteins diminished activation by VP16-NCoR-ID by more than 50%, reflecting the reduced interaction with NCoR-ID. The same analyses were performed with another CoR (VP16-SMRT), and a nearly identical pattern of results was obtained, except that the degree of stimulation was not as great as that with VP16-NCoR (data not shown).



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  		Figure 3. Interactions of Gal4-TR mutant proteins with NCoR in mammalian two-hybrid assays. A, Gal4 expression plasmids (25 ng) were cotransfected into TSA-201 cells with VP16-NCoR-ID or a VP16 empty vector (250 ng) together with 100 ng of the Gal4-responsive reporter gene, UAS-TK-Luc. Cells were incubated in the absence of T3. Results are the mean ±SD from three independent experiments. B, The silencing activity of the Gal4-TR mutant fusion proteins was assessed in the absence of T3 and without the addition of VP16-NCoR-ID. C, Correlation between silencing activity and the in vivo interaction with VP16-NCoR-ID. The silencing activity is expressed as fold repression (data from B) of Gal4-TR fusion proteins and is plotted vs. fold stimulation mediated by the interaction between Gal4-TR and VP16-NCoR-ID fusion proteins (data from A). Data in A and B are the mean ± SD from three independent experiments.

 
Silencing by TRß mutants correlates with NCoR interactions
The silencing activities of the wild-type and RTH mutants were also examined using the Gal4-TR constructs (16). Wild-type Gal4-TRß strongly silenced expression (75% repression) from UAS-TK-Luc compared with Gal4-DBD alone (Fig. 3BGo). Potent silencing was also seen with each of the three RTH mutants with a rank order of Gal4-P453X ~ P453H > G345R. Insertion of the P214R mutation into the background of each of Gal4-TR fusion proteins reduced silencing activity by about 50%, such that the activity remained proportionate to the original silencing activity of the RTH mutant concerned.

The relationship between the silencing activity and the intensity of interaction with NCoR was determined by comparing the silencing activity (fold repression; Fig. 3BGo) with the degree of stimulation mediated by Gal4-TR and VP16-NCoR-ID interactions (data from Fig. 3AGo). This analysis is shown in Fig. 3CGo and reveals a significant positive correlation (r = 0.937; P < 0.001), confirming the idea that interaction with CoRs is required for silencing.

Functional properties of TRß mutants with respect to positively regulated genes
The functional properties of the mutant TRs were examined using two different types of positively regulated T3-responsive reporter genes (PAL-TK-Luc and DR4-SV40-Luc). When wild-type or RTH mutant TRs were transfected into TSA-201 cells together with the positively regulated genes, pronounced silencing was observed in the unliganded state (without T3) for both types of reporter genes (Fig. 4Go, A and C). In contrast, little or no silencing was observed with the P214R mutant. Insertion of P214R mutation into the background of the RTH mutants markedly reduced the silencing by these otherwise potent mutants.



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  		Figure 4. The functional properties of mutant TRs using positively regulated genes. A and B, Positively regulated PAL-TK-Luc. Wild-type TRß or mutant expression plasmids (10 ng) were transfected into TSA-201 cells together with 250 ng of the reporter gene PAL-TK-Luc. C and D, Positively regulated DR4-SV40-Luc. Wild-type TRß or mutant expression plasmids (100 ng) were transfected into TSA-201 cells together with 10 ng of the reporter gene DR4-SV40-Luc. Cells were incubated in the absence or presence of 1 nM T3. Results are the mean ± SD from three independent experiments.

 
In the presence of 1 nM T3, wild-type TRß and the P214R mutant each stimulated the reporter genes (Fig. 4Go, B and D). However, the fold stimulation by TRß (38-fold) was much greater than that seen with the P214R mutation (18-fold; Fig. 4BGo), reflecting its more pronounced silencing activity. In contrast, each of the RTH mutants and their double mutants with P214R showed no trans-activation, consistent with their reduced ability to bind to T3 (41).

The level of expression of the RTH double mutants was examined using gel mobility shift assays of extracts from transfected TSA-201 cells to confirm that the loss of silencing and trans-activation was not due to the decreased expression (Fig. 5Go). Using a DR4-binding site, comparable amounts of binding activity were found in cells transfected with wild-type or each of the mutant TRs. Preliminary experiments showed that the binding activity migrated predominantly as a heterodimer complex (data not shown), and subsequent experiments were performed in the presence of RXR to assure detection of all TR protein. No binding was seen in extracts from mock-transfected cells. A similar amount of binding activity was seen with the single RTH and P214R mutants (data not shown).



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  		Figure 5. Expression of wild-type and mutant TRs in transiently transfected TSA-201 cells. DNA binding of transfected ß1 wild-type or mutant receptors was analyzed using 32P-labeled DR4-TRE in nondenaturing gel electrophoresis. The labeled DR4 probe was incubated with whole cell lysates from transiently transfected TSA-201 cells and in vitro translated RXR{alpha} as indicated. RL-TRß denotes in vitro translated TRß1.

 
Functional properties of the TRß mutants with respect to negatively regulated genes
In contrast to the silencing of positively regulated genes by unliganded TR, negatively regulated genes were transcriptionally induced by unliganded TR and repressed upon the addition of T3 (6, 8, 42). The P214R CoR mutation was shown to diminish the basal activation of negatively regulated promoters by unliganded TR, suggesting that CoRs play a role in this process (16). The functional properties of the RTH mutants were examined using two negatively regulated promoters (TSH{alpha}-Luc and TSHß-Luc; Fig. 6Go). Wild-type unliganded TRß induced basal stimulation of both negatively regulated promoters (Fig. 6Go, A and C). The P453X RTH mutant caused even greater basal activation than the wild-type receptor, whereas the effects of the P453H and G345R mutants were less pronounced. In contrast, the P214R mutant showed loss of basal activation. Introduction of the P214R mutation into the background of the RTH mutants decreased their basal activation. This effect was most prominent with the P453X/P214R double mutant. In the presence of 1 nM T3, all RTH mutants and their double mutants with P214R exhibited no T3-dependent repression, consistent with their inability to bind T3 at this dose (Fig. 6Go, B and D). The degree of T3-induced repression by the P214R mutant was reduced, primarily reflecting the loss of basal activity.



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  		Figure 6. The functional properties of mutant TRs using negatively regulated genes. A and B, Negatively regulated TSH{alpha}-Luc. Wild-type TRß or mutant expression plasmids (100 ng) were transfected into TSA-201 cells together with 100 ng of the reporter gene TSH{alpha}-Luc. C and D, Negatively regulated TSHß-Luc. Wild-type TRß or mutant expression plasmids (100 ng) were transfected into TSA-201 cells together with 100 ng of the reporter gene TSHß-Luc. Cells were incubated in the absence or presence of 1 nM T3. Results are the mean ± SD from three independent experiments.

 
The P214R CoR mutation abrogates the dominant negative activity of RTH mutant receptors for both positively and negatively regulated genes
Having established the functional properties of the individual receptor mutants, their dominant negative activities were determined in the presence of wild-type receptor (Fig. 7Go). Because each of the RTH mutants (and their double mutants with P214R) exhibited no trans-activation or T3-dependent repression in the presence of 1 nM T3 (Figs. 4Go and 6Go), this concentration was used to examine their dominant negative activities. Although the RTH mutants partially block the activity of the wild-type receptor at a 1:1 ratio (41) (data not shown), a 1:5 ratio of wild-type to mutant receptors was used to more clearly illustrate the dominant negative properties of the mutant receptors.



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  		Figure 7. Effect of the P214R CoR mutation on the dominant negative activity of RTH receptor mutants. A, Mutant TR expression plasmids (25 ng) were cotransfected with 5 ng wild-type TRß into TSA-201 cells together with 250 ng of the reporter gene PAL-TK-Luc. B, Mutant TR expression plasmids (250 ng) were cotransfected with 50 ng wild-type TRß into TSA-201 cells together with 10 ng of the reporter gene DR4-SV40-Luc. C and D, Mutant TR expression plasmids (250 ng) were cotransfected with 50 ng wild-type TRß into TSA-201 cells together with 100 ng of the reporter gene TSH{alpha}-Luc (C) or TSHß-Luc (D). Cells were incubated in the absence or presence of 1 nM T3. Results are the mean ± SD from three independent experiments.

 
T3-dependent activation of PAL-TK-Luc by wild-type receptor (34-fold) was inhibited by cotransfection with P453X, P453H, and G345R mutants (T3 stimulation = 3-, 7-, and 9-fold, respectively; Fig. 7AGo). In contrast, cotransfection of the CoR mutant (P214R) did not inhibit wild-type receptor function (40-fold), confirming that it does not exhibit dominant negative properties. Insertion of the CoR mutation into the RTH mutants (P453X/P214R, P453H/P214R, and G345R/P214R) greatly diminished the dominant negative activity of the RTH mutants (T3 stimulation = 23-, 26-, and 34-fold, respectively). Similar results were seen with the DR4-driven promoter (Fig. 7BGo). This effect of the P214R mutation reflects loss of silencing as well as decreased inhibition of T3 stimulation.

With the negatively regulated TSH{alpha} promoter, T3-induced repression by the wild-type receptor (3.3-fold) was blocked by cotransfection with the P453X, P453H, and G345R RTH mutants (T3 repression = 1.5-, 1.7-, and 1.7-fold, respectively; Fig. 7CGo). Insertion of the P214R CoR mutant into each of the RTH mutants (P453X/P214R, P453H/P214R, and G345R/P214R) impaired their dominant negative activities (T3 repression = 2.6-, 2.8-, and 2.9-fold, respectively). Similar effects were seen with the negatively regulated TSHß promoter (Fig. 7DGo). These results suggest that the interaction with CoRs is critical for the dominant negative effects of RTH mutants with respect to both positively and negatively regulated genes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we provide several lines of evidence that nuclear CoRs play a role in the syndrome of resistance to thyroid hormone. The RTH mutants retain the ability to interact with CoRs, as revealed by gel shift assays and mammalian two-hybrid interaction assays. Moreover, because the RTH mutants are defective in T3 binding, they are unable to dissociate CoR, as normally occurs with the wild-type TR. The dominant negative activity of the RTH mutants was remarkably dependent upon interactions with CoRs; insertion of a mutation (P214R) that reduces CoR binding nearly eliminated dominant negative activity. Finally, the role of CoRs in the dominant negative activity of RTH mutants is apparent not only for genes that are positively regulated by T3, but also for negatively regulated genes. These findings indicate that CoR are involved in the pathogenesis of RTH and support an important role for CoR in the control of negatively regulated genes.

The molecular pathogenesis of RTH has been studied rather extensively (for review, see Ref.25). Nevertheless, a number of questions remain unresolved. Early experiments documented that the mutant receptors function in a dominant negative manner to inhibit the actions of wild-type TR (19, 22, 23). RTH mutants, which only occur naturally in the TRß gene (43), were shown to inhibit the activity of either TR{alpha} or TRß, and the dominant negative activity is exerted with respect to both positively and negatively regulated reporter genes (23). Mutations in the DBD of the RTH mutants eliminate their dominant negative activity, suggesting that inhibition requires interactions with DNA target sites (27). In addition, mutations that selectively impair heterodimerization with RXR also eliminate the dominant negative activity of RTH mutants, probably because the RXR-TR heterodimer facilitates DNA binding to target genes (20, 33, 44). TR homodimers may play a role in the dominant negative properties of the RTH mutants. T3 dissociates TR homodimers from DNA (45), but RTH mutants lack this property because of decreased T3 binding affinity (46). Thus, inactive TR homodimers may retain preferential binding to T3-responsive target genes. Transfection experiments using homodimer-selective response elements support a role for inhibitory homodimer complexes (47). Consistent with this idea, certain naturally occurring RTH mutants that are selectively defective in homodimerization tend to have reduced dominant negative activity (20, 48, 49, 50). In this regard, it is noteworthy that introduction of the P214R mutation appears to decrease the relative amount of homodimer, at least for the limited number of receptor mutants studied. This or other conformational effects of the P214R mutation may, therefore, have effects other than decreasing interactions with NCoR. Several studies demonstrate that RTH mutations are associated with a broad phenotypic spectrum (26, 51). It remains unclear whether the severity of the clinical phenotype correlates with the potency of dominant negative inhibition in transient expression assays, although most studies suggest such a relationship (15, 19, 20, 41, 49, 50, 52, 53).

The recent discovery of CoRs that interact with the TR (9, 11) offers yet another pathway that may impact upon the molecular pathogenesis of RTH. In earlier studies, it was noted that the TR contains a transferable "silencing domain" in its carboxyl-terminus (54), and that RTH mutants retain this silencing function in a constitutive manner that is not reversed by T3 (55). This finding raised the possibility that in addition to competition for wild-type TR interactions with target genes, RTH mutants might be capable of actively repressing target genes. Subsequent studies confirmed basal silencing of positively regulated promoters by RTH mutants (56). The identification of CoRs and their interaction domains within the TR (9, 10, 11, 14) has allowed their roles in RTH to be assessed more directly. Our studies confirm and extend a recent report by Yoh et al. (15) that examines the interactions of CoRs with RTH mutants. These investigators found that RTH mutants bound to glutathione-S-transferase-SMRT in protein interaction assays and that they failed to dissociate normally in the presence of T3 (15). As in our experiments, insertion of TR mutations that disrupt interactions with SMRT greatly reduced the dominant negative activity of the RTH mutants.

There is a striking correlation between the degree of transcriptional silencing activity and the strength of TR interactions with CoRs as assessed by mammalian two-hybrid assays. The correlation was observed with NCoR, and in experiments not shown, very similar results were obtained using SMRT, except that there was a smaller maximal increase by VP16-SMRT than by VP16-NCoR. The correlation was also seen when either DR4 or palindromic response elements were used to assess silencing by native receptors rather than the Gal4-TR fusion proteins (data not shown). This observation raises the possibility that the strength of interaction with CoRs may account in large measure for the potency of dominant negative activity. For example, in the current series, the rank order for NCoR interaction and silencing (P453X > P453H > G345R) corresponds to the dominant negative potencies for these mutants (41). Yoh et al. (15) also observed differences in the interactions of various RTH mutants with glutathione-S-transferase-SMRT and suggested that variability in CoR interactions might account for differences in dominant negative potency. Ultimately, a large array of mutants will need to be examined to verify this intriguing concept. In addition, it remains challenging to correlate the degree of hormone resistance in different tissues in vivo with the dominant negative potency observed in transfected cells (53).

An important observation in this report is the finding that CoRs play a role in the dominant negative activity of RTH mutants with respect to both positively and negatively regulated genes. A model for CoR action is presented in Fig. 8Go. In the case of positively regulated genes, the role of the CoR fits well with existing concepts concerning the pathophysiology of RTH. As described above, the CoR interacts with the RTH mutant and induces transcriptional silencing of the target gene. In cases in which T3 binding is decreased, T3 is unable to dissociate the CoR, and to the extent that mutant TR homodimers are bound, these complexes will also remain intact. In addition to silencing, the RTH mutants are incapable of recruiting coactivators to induce transcriptional activation. Finally, the RTH mutants compete with wild-type TRs for access to target genes.



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  		Figure 8. Model depicting the role of CoRs in the dominant negative activity of thyroid hormone resistance mutants. The prevailing model for the effects of TR on positively regulated genes is shown at the top of A. The TR partner is RXR, but TR-TR homodimers also probably play a role. In the absence of T3, CoRs bind to TR and mediate silencing. In the presence of T3, CoRs dissociate, and coactivators bind to TR, resulting in relief of silencing and transcriptional activation. In the case of the thyroid hormone resistance mutant, which does not bind to T3, a suppressive complex is formed with CoR, and this complex competes for wild-type receptor binding to DNA to block transcriptional activation (bottom of A). A model for negatively regulated genes is shown at the top of B, although the roles of TR and CoR are less well defined. TRs may act indirectly on negatively regulated genes through protein-protein interactions. In the absence of T3, CoRs may bind to TR and stimulate basal expression, perhaps because they have been withdrawn from other promoter targets. In the presence of T3, CoRs dissociate, and coactivators bind to TR, resulting in transcriptional repression. In the case of the thyroid hormone resistance mutant, TR forms a complex with CoRs to activate the promoter, and it competes for the ability of the wild-type TR to gain access to the target gene to prevent T3-dependent transcriptional repression (bottom of B). GTFs, General transcription factors.

 
The situation with negatively regulated genes is less well understood, primarily because the mechanisms for T3-induced repression have not been elucidated. Recently, we showed that in conjunction with unliganded TR, CoRs induce basal activation of negatively regulated promoters (16). This conclusion was based in large part upon the loss of basal activation by TR mutations (P214R) that eliminated interactions with CoRs. In addition, excess exogenous NCoR or SMRT increased basal activity further and required interactions with TR, supporting the idea that CoRs act as coactivators for this class of promoters. It remains possible, however, that TRs act to partition NCoRs, such that a repressive activity on promoter-bound proteins is decreased. Thus, in the context of RTH mutants, we propose that this activating function of CoRs is increased, in part because of enhanced interactions with mutant receptors. Even though the mechanisms of T3-induced repression remain obscure, the inability of T3 to dissociate the CoR from RTH mutants would retain the negatively regulated promoters in a constitutively activated state unless the mutant receptor is displaced by wild-type TR. In this manner, genes such as TSH{alpha} and TSHß would be stimulated by RTH mutants, accounting in part for one of the hallmark features of the syndrome.

Many questions remain unanswered. Can CoRs account for some of the tissue-specific differences in hormone resistance in RTH? If the concentration or composition of CoRs varies among tissues, our results would predict tissue-specific differences in responses to T3. Another intriguing question is why dominant negative activity is so markedly affected by CoR interactions. Because reversal of silencing accounts 50% or less of the fold stimulation by T3 in most experimental circumstances, one might have anticipated a less marked effect of the P214R CoR mutation on dominant negative activity. It is possible, for example, that CoRs also stabilize mutant receptor complexes on DNA. In the case of negatively regulated promoters, the nature of the TR complex is unknown, and the mechanism by which CoRs cause activation is not understood. Further studies will be of interest to address these and other issues related to the role of CoRs in the syndrome of RTH.


   Acknowledgments
 
We are grateful to L. Madison, P. Kopp, T. Nagaya, K. Kitajima, and M. G. Rosenfeld for providing plasmids.


   Footnotes
 
1 This work was supported by NIH Grant DK-42144. Back

Received July 2, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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