An Inhibitory Region of the DNA-Binding Domain of Thyroid Hormone Receptor Blocks Hormone-Dependent Transactivation

Ying Liu1, Akira Takeshita, Takashi Nagaya, Aria Baniahmad, William W. Chin and Paul M. Yen1

Division of Genetics, Department of Medicine Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts 02115
Department of Endocrinology and Metabolism (T.N.) Research Institute of Environmental Medicine Nagoya University Nagoya, Japan
Genetisches Institut (A.B.) Justus-Liebig-Universitat D-35392, Giessen, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor ß ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-ß (TRß), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRß derepressed transcription to basal level. Using this system and a battery of TRß mutants, we found that TRß/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRß ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRß DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRß from achieving transcriptional activation above basal level in this chimeric receptor system.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone receptors (TRs) are nuclear hormone receptors that regulate transcription of target genes by binding to thyroid hormone response elements (TREs) in their promoter regions. As such, they function as ligand-regulatable transcription factors which can activate transcription of positively-regulated target genes in the presence of T3. TRs can bind to TREs as homodimers and heterodimers, particularly with retinoid X receptors (RXRs). On the basis of DNA binding in the presence of T3, cotransfection studies with TR mutants, in vitro transciption studies, and yeast systems that do not contain endogenous TR and RXR, TR/RXR heterodimers likely are the transcriptionally active complexes involved in T3-mediated transcriptional activation for most TREs (1, 2, 3).

Interestingly, for several positively regulated target genes, TRs also can repress basal transcription in the absence of ligand. Addition of T3 relieves this basal repression and activates transcription above basal level. We and others previously have shown that binding of unliganded TRs to TREs, several groups have isolated and cloned the cDNAs of proteins (corepressors) that interact with TRs in a ligand-dependent manner such as N-CoR (nuclear receptor corepressor) and SMRT (8, 9, 10, 11). In particular, these proteins interact with unliganded, but not liganded, TRs. The subregion of TR that interacts with corepressors appears to be located in the hinge region between the DNA- and ligand-binding domains (LBDs) (8, 9). Functional studies suggest that these putative corepressors can mediate basal repression of transcription when associated with TRs.

In addition to corepressors, there are several putative coactivators such as steroid receptor-coactivator-1 (SRC-1), transcriptional intermediary factor II (TIFII)/GRIP1, P300/CBP cointegrator-associated protein (p/CIP), and the recently described receptor-associated coactivator-RAC3/ACTR/AIB1/thyroid hormone receptor activator molecule-1 (TRAM-1) that interact with TRs or other members of the nuclear hormone receptor family that may be important in mediating ligand-dependent transcriptional activation for these receptors (12–19a). In contrast to corepressors, these proteins selectively interact with the liganded, rather than unliganded, nuclear hormone receptors. Mutations and deletions of a highly conserved section of the extreme carboxy-terminal region of TRs and other nuclear hormone receptors [activation function (AF)-2 domain] have shown that this region is critical for mediating ligand-dependent transcription and in some cases, interactions with putative coactivators (20, 21, 22, 23). It is possible that this region may interact directly with coactivators or may exert allosteric effects on TR conformation that may influence TR interaction with coactivators. The role of the AF-2 domain in modulating basal repression and derepression is currently not well characterized.

Presently, little is known about the potential role of the DNA-binding region on transcriptional activity by TRs. It appears that steric effects mediated by DNA binding may affect retinoic acid receptor/RXR and TR/RXR interactions with corepressors and/or coactivators (24). Additionally, mutations in the first zinc finger of TRß still can allow DNA binding by abrogating transcriptional activity (25). However, the intrinsic role of the DNA-binding domain (DBD) on transcription has not been investigated fully.

The GAL4 chimeric receptor system has been successfully employed recently to study the specific role(s) of TR subregions in transcriptional activation and protein-protein interactions (23, 26, 27). Using this system, and a battery of TRß mutants (Fig. 1Go, A and B), we have found that TRß/RXR heterodimers may have different transcriptional activity depending upon which heterodimer partner binds DNA. We also found that TRß/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, a mutation in the TR AF-2 region did not alter basal repression but was unable to mediate derepression, suggesting that interactions with coactivators may be important for derepression. Surprisingly, the TRß LBD not only could repress basal transcription but also could transactivate above basal level in the presence of T3. Deletion and domain swap of different regions of TRß suggest that there is a region in the second zinc finger of TRß that may inhibit ligand-dependent transcriptional activation by the full-length receptor by an allosteric mechanism.



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Figure 1. Chimeric Receptor System and TRß Mutants

A, Model depicting chimeric receptor system. B, Summary of TRß and TRß mutants. Receptors were translated in vitro according to manufacturer’s instructions (Promega) and then analyzed for [125I]T3 binding or DNA binding to the F2 TRE as previously described (22, 40, 42, 50). Data are summary of previously published and new results (40, 42, 50). T3-dependent transactivation by these receptors was measured via a luciferase reporter plasmid containing F2 TRE in cotransfection experiments (40).

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We first examined the transcriptional activity of GAL4RXR and GAL4TRß in the absence or presence of their cognate ligands (Fig. 2Go). 9-cis-RA stimulated GAL4RXR-mediated transcriptional activity 25-fold, and T3 stimulated GAL4TRß transcriptional activity greater than 15-fold. Interestingly, 9-cis RA also could stimulate GAL4TRß-mediated transcription presumably via heterodimerization with endogenous RXR. We previously have observed that RXRß is predominantly expressed in CV-1 cells using isoform-specific antibodies (Ref. 28 and P. M. Yen, unpublished results). Cotransfection of RXRß did not further augment this 9-cis-RA effect on GAL4-TR-mediated transcripton. In contrast, T3 did not affect GAL4RXR-mediated transcription presumably because CV-1 cells contain little or no endogenous TRs.



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Figure 2. Transcriptional Transactivation by GAL4 RXR and GAL4TRß in the Presence or Absence of Ligand and/or Heterodimer Partners

hTRß or mRXRß expression vector (0.1 µg) was cotransfected in CV-1 cells in the absence or presence of 10-6 M T3 or 9-cis-RA for 24 h as indicated. In these experiments, treated cells were then harvested and luciferase was measured. Luciferase activity was normalized to ß-galactosidase activity and then calculated as fold basal luciferase activity with 1-fold basal activity defined as the luciferase activity with control GAL4 and pcDNA vector alone in the absence of ligand. pcDNA vector was added to some samples so that each sample had the same amount of total expression vector. Each value represents the mean of four samples, and bars denote SD of the mean.

 
In the absence of ligand, GAL4TRß, but not GAL4RXR, repressed transcription to less than 20% of basal level. When TRß was coexpressed with GAL4RXR, similar basal repression occurred in the absence of ligand. Thus, GAL4TRß/RXR and GAL4RXR/TRß complexes are both able to mediate basal repression. However, in contrast to GAL4TRß alone and GAL4TRß and RXRß, which activated transcription in the presence of T3, GAL4RXR and TRß derepressed basal repression in a T3-dependent manner, but did not activate transcription significantly above basal level. Additionally, GAL4RXR and TRß blocked 9-cis-RA-mediated transcription in the presence or absence of T3 similar to previously reported findings (29, 30). Thus, in this chimeric system, TRß had different effects on RXR-mediated transcription depending on the absence or presence of cognate ligand for either TR or RXR.

It has been difficult to separate derepression from transcriptional activation in previous studies using conventional cotransfection studies employing full-length receptors and TRE-containing reporters. The previous findings thus prompted us to study in greater detail the mechanisms of GAL4RXR- and TRß-mediated repression and derepression. We first examined the role of heterodimerization on repression and derepression by using TRß mutants in the ninth heptad regions, which selectively formed homo- or heterodimers on electrophoretic mobility shift assays (Fig. 1Go). In these studies the mutants were co-transfected with GAL4 vector as a control or GAL4RXR vector with the upstream activating sequence (UAS)-reporter. As seen in Fig. 3Go, the hTRß and GAL4 samples had weak basal repression and derepression in the presence of T3, suggesting there may be a weak TRE present in the expression vector. When cotransfected with GAL4RXR, hTRß repressed basal transcription to 10% basal level and derepressed in the presence of T3. Heterodimer-specific mutant R429Q behaved similar to wild-type TRß, but homodimer-preferential mutant L428R was unable to mediate basal repression and derepression. G345R, a natural mutant from a patient with resistance to thyroid homone, exhibited constitutive basal repression. TRAHTm, a TRß that contains three amino acid substitutions in the hinge region and had decreased affinity with the putative corepressor, N-CoR (nuclear receptor corepressor), showed reduced basal represssion in the absence of ligand (8). These findings suggest that heterodimerization and an intact hinge region are important for basal repression in this system, and derepression depends on T3 binding to TR.



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Figure 3. Repression and Derepression by GAL4RXR and hTRß or TRß Mutants for Homodimerization (L428R), Heterodimerization (R429Q), T3 Binding (G345R), and Basal Repression (TRAHTM)

hTRß or hTRß mutant vectors (0.1 µg), GAL4 or GAL4RXR expression vectors (0.1 µg), UAS-containing reporter plasmid (1.7 µg), and ß-galactosidase control vector (1.0 µg) were cotransfected in CV-1 cells in the absence or presence of 10–6 M T3 for 24 h as indicated. In these experiments, treated cells were then harvested and luciferase was measured. Luciferase activity was normalized to ß-galactosidase activity and then calculated as fold basal luciferase activity with control GAL4 and pcDNA vector alone in the absence of ligand. pcDNA vector was added to some samples so that each sample had same amount of total expression vector. Each value represents the mean of three to six samples, and bars denote SD of the mean.

 
We next examined the effect of point mutations in the AF-2 region located in the extreme carboxy-terminal region of TRß on repression and derepression (Fig. 4Go). Previously, Chatterjee and co-workers (22) showed that these mutations markedly reduced transcriptional activation. As seen in Fig. 2Go, both E457A and E457D were able to mediate basal repression in the absence of ligand. However, in the presence of T3, E457D, which contains a more conservative amino acid substitution, derepressed to basal transcription level, but E457A did not. These findings suggest that mutations in the AF-2 region not only affect transcriptional activation but also may affect derepression and thus lead to constitutive basal repression.



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Figure 4. Repression, Derepression, and Transcriptional Activation by GAL4RXR and rTRß, AF-2 Domain rTRß Mutants (E457A, E457D), or LBD Mutants

rTRß, AF-2 domain rTRß mutants, or rTRß LBD vectors (0.1 µg), GAL4 or GAL4RXR expression vectors (0.1 µg), UAS-containing reporter plasmid (1.7 µg), and ß-galactosidase control vector (1.0 µg) were cotransfected in CV-1 cells in the absence or presence of 10-6 M T3 for 24 h as indicated. In these experiments, treated cells were then harvested and luciferase was measured. Luciferase activity was normalized to ß-galactosidase activity and then calculated as fold basal luciferase activity with 1-fold basal activity defined as the luciferase activity with control GAL4 and pcDNA vector alone in the absence of ligand. pcDNA vector was added to some samples so that each sample had same amount of total expression vector. Each value represents the mean of four samples, and bars denote SD of the mean.

 
We also examined the effects of the TR ligand binding domain in this system (Fig. 4Go). TR-LBD (which also contains the hinge region for nuclear localization and putative corepressor interaction sites) was able to mediate basal repression in the absence of ligand. Surprisingly, LBD not only derepressed basal repression, but also stimulated transcription greater than 10-fold over basal level. The LBDs that contain AF-2 mutations were able to repress basal transcription and had markedly impaired transcriptional activation.

These findings with full-length TRß and TR-LBD suggested that there may be an inhibitory region for T3-mediated transcriptional activation that was located in either the amino-terminal region or DBD of TRß. We thus studied the effects on basal repression and transcriptional activation using a mutant TRß in which the amino-terminal region has been deleted, TRß-{Delta}N (Fig. 5Go). The receptor had decreased basal repression but exhibited no transcriptional activation in the presence of T3. Similar effects by TRß-{Delta}N on basal repression recently have been reported by Hollenberg et al. (31). Additionally, TR{alpha}, which contains a 40-amino acid amino-terminal region that does not have homology with TRß was unable to activate transcription in the presence of T3. Taken together, these results suggest that the amino-terminal region is unlikely to be mediating inhibition of transcriptional activation by full-length TRß.



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Figure 5. Repression, Derepression, and Transcriptional Activation by GAL4RXR and rTR{alpha}, TRß, and TRß{Delta}N

rRT{alpha}, rTRß, and TRß{delta}N vectors (0.1 µg), GAL4 or GAL4RXR expression vectors (0.1 µg), UAS-containing reporter plasmid (1.7 µg), and ß-galactosidase control vector (0.1 µg) were cotransfected in CV-1 cells in the absence or presence of 10-6 M T3 for 24 h as indicated. In these experiments, treated cells were then harvested and luciferase measured. Luciferase activity was normalized to ß-galactosidase activity and then calculated as fold basal luciferase activity with 1-fold basal activity defined as the luciferase activity with control of GAL4 and pcDNA vector alone in the absence of ligand. pcDNA vector was added to some samples so that each sample had same amount of total expression vector. Each value represents the mean of four samples, and bars denote SD of the mean.

 
We next used a chimeric TR, TRß-TGT, in which the DBD has been swapped with the corresponding domain from the human glucocorticoid receptor (GR) and observed a 4-fold activation above basal transcription level in the presence of T3 (Fig. 6AGo). These findings suggested that there may be a subregion within the DBD that may be involved in inhibiting transcriptional activation. Accordingly, we used TRß mutants in which each zinc finger was swapped with the corresponding zinc finger of GR (TRß-TG and TRß-GT) and studied their abilities to activate transcription (Fig. 6BGo). TRß-TG, but not TRß-GT, was able to able to stimulate transcription greater than 10-fold in the presence of T3 suggesting that the inhibitory region was located in or near the second zinc finger.



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Figure 6. Transcriptional Activation by Zinc Finger Swap Mutants (TRß-TGT, TRß-TG, and TRß-GT)

TRß-TGT in pRSV, TRß-TG and TRß vectors (0.1 µg), GAL4 or GAL 4RXR expression vectors (0.1 µg), UAS-containing vector plasmid (1.7 µg), and ß-galactosidase control vector (1.0 µg) were cotransfected in CV-1 cells in the absence or presence of 10-6 M T3 for 24 h as indicated. In these experiments, treated cells were then harvested and luciferase was measured. Luciferase activity was normalized to ß-galactosidase activity and defined as the luciferase activity with control GAL4 and pcDNA or pRSV vector alone in the absence of ligand. pcDNA or pRSV vector was added to some samples so that each sample had the same amount of total expression vector. Each value represents the mean of four samples, and bars denote SD of the mean. A, TRß-TGT; B, TRß-TG and TRß-GT.

 
The mechanism of this inhibition could be due to allosteric changes in the TR DBD that modulate the conformation of TR and reduce its affinity for coactivators. Alternatively, it could be due to cellular inhibitors that interact with this region of TR and reduce full-length TR’s affinity for coactivators. To determine whether there may be a cellular inhibitor blocking the transcriptional activity of full-length TRß, we cotransfected a 3-fold excess of L428R expression vector with TRß and GAL4RXR expression vectors to examine whether it would titrate out a putative inhibitor(s) (Fig. 7Go). This mutant forms heterodimers poorly and did not repress basal transcription or transactivate (Figs. 1Go and 3Go). As seen in Fig. 7Go, L428R was unable to enhance transcriptional activation by TRß, suggesting that it was not titrating out an inhibitor. L428R also did not affect the transcriptional activation by LBD. Even addition of a 20-fold excess of L428R had no significant effect on transcriptional activity by TRß or LBD (data not shown). Similar results also were obtained when we used a mutant TR containing only the TRß DBD and hinge region in this system (data not shown). These results argue against a titratable inhibitor interfering with the transcriptional activation of TRß.



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Figure 7. Titration of a Putative Inhibitor by L428R Mutant

TRß vector (0.1 µg), L428R mutant (0.3 µg), GAL4, or GAL4RXR expression vectors (0.1 µg), UAS-containing reporter plasmid (1.7 µg), and ß-galactosidase control vector (1.0 µg) were cotransfected in CV-1 cells in the absence or presence of 10-6 M T3 for 24 h as indicated. In these experiments, treated cells were then harvested and luciferase measured. Luciferase activity was normalized to ß-galactosidase activity and then calculated as fold basal luciferase activity with 1-fold basal activity defined as the luciferase activity with control GAL4 and pcDNA vector alone in the absence of ligand. pcDNA vector was added to some samples so that each sample had same amount of total expression vector. Each value represents the mean of three samples, and bars denote SD of the mean.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have used a GAL4 chimeric receptor system to study the mechanisms of repression, derepression, and transcriptional activation for TRs. We observed some common features as well as differences depending on whether GAL4TRß or GAL4RXR was used. GAL4TRß, but not GAL4RXR, repressed basal transcription in the absence of ligand. However, when these chimeric receptors were cotransfected with their corresponding heterodimer partner, both repressed basal transcription. GAL4RXR mediated 9-cis-retinoic acid-dependent transcriptional activation; however, similar to previous studies, TRß partially blocked the amount of 9-cis-RA-dependent transcriptional activation by GAL4RXR (29, 30).

9-cis-RA also activated transcription by GAL4TRß (presumably via endogenous RXR heterodimerized with GAL4TRß) and GAL4TRß and RXR. The amount of transcriptional activation by 9-cis-RA was similar to that observed for the reciprocal complex of GAL4RXR and TRß. GAL4TRß or GAL4TRß and RXRß activated transcription in the presence of T3. In contrast, GAL4RXR and TRß only derepressed transcription in the presence of T3. These findings suggest that there may be different effects on derepression and transcriptional activation depending on which heterodimer partner is bound to DNA and/or conformational differences between GAL4RXR/TRß and GAL4TRß/RXR heterodimer complexes.

The observation that GAL4RXR and TRß repressed and derepressed in the absence or presence of T3 suggested that this system would enable us to study these two functions of the TR apart from transcriptional activation. Studies using natural and artificial TRß mutants showed that heterodimerization and an intact hinge region that can interact with corepressors such as N-CoR are necessary for basal repression. T3 binding is critical for derepression as a natural TRß mutant that has minimal T3 binding exhibited constitutive basal repression in the presence of T3.

Our studies with AF-2 mutants suggest that mutations in this region do not significantly affect basal repression. However, one of the mutants (E457A) was unable to derepress in the presence of T3. This mutant has similar hormone binding affinity as wild-type TRß (22, 32). These findings suggest that mutations in the AF-2 region may modulate derepression. It is possible that binding of coactivators may be necessary for release of corepressors from TR, and the equilibrium between corepressor- and coactivator-bound TR determines the amount of basal repression, derepression, and transcriptonal activation. Recent work by Baniahmad et al. (23) and Schulman et al. (27) also support this possibility.

TR-LBD, which contains the hinge region to ensure nuclear localization, was able to repress basal transcription in this system. Surprisingly, it not only derepressed basal transcription but also was able to activate transcription in the presence of T3. When the AF-2 region LBDs were used, there was little or no trancriptional activation above basal level, confirming the critical role of this region for transcriptional activation. Our data suggest the difference between the transcriptional activity of full-length TRß and TR-LBD is due to inhibition of transcriptional activation by a subregion near or within the second zinc finger of the DBD. It is not known whether this subregion inhibits transcription by TR/RXR heterodimers bound to TREs. Indeed, when LBD is cotransfected with TRE-containing reporters, we and others did not observe either basal repression or transcriptional activation. However, Forman et al. (33) showed that LBD is unable to bind as a dimer with TR or RXR to TREs suggesting that it may not be possible to detect this inhibitory function on conventional TRE-containing reporters.

The mechanism of this inhibition may be due to cellular inhibitors that interact with the TR DBD or to allosteric changes in the TR DBD that modulate the conformation of TR and reduce its affinity for coactivators. Casanova et al. (34) have provided evidence for a cellular inhibitor that interacts with unliganded TR in a region that involves the ninth heptad region of the cTR{alpha} LBD and is released when T3 binds to TR. Burris et al. (35) have shown that a TR-interacting protein identified by two-hybrid screening inhibits TR-mediated transcription by binding to the TR hinge and amino-terminal region of the LBD. In contrast, our titration experiments suggest that allosteric changes induced by the DBD, rather than cellular inhibitors, likely account for the difference in transcriptional activation. Furthermore, cotransfection studies in P19 embryonal carcinoma cells exhibited similar differences among the transcriptional activities of TRß, TR LBD, and TRß-TG, as observed in CV-1 cells, and thus argue against a cell-specific inhibitor. Finally, we have performed far-Western blots of nuclear extracts using 32P-labeled glutathione-S-transferase (GST) rat TRß LBD and full-length rat TRß (32). Similar to recent work by Fondell and Roeder using a coimmunoprecipitation assay, we observed that the TRs can interact with several different nuclear proteins in a T3-dependent manner (36); however, GST-rat TRß LBD interacted much more strongly with this group of nuclear proteins than GST full-length rat TRß (A. Takeshita, unpublished data). Taken together, these findings strongly suggest that the DBD may modulate the conformation of the AF-2 region of TR. Recently, the crystal structure of ligand-bound TR{alpha} LBD was solved (37). Our data raise the possiblity that the coordinates of certain subregions within the LBD crystal structure, such as the AF-2 domain, may be different for the full-length receptor TR than the LBD and should be interpreted with caution. Crystal structures of TR containing both the DBD and LBD will be helpful in resolving this issue.

In summary, allosteric changes by other regions of the receptor, protein-protein interactions (as seen by the differences between T3-mediated transactivation by GALRXR/TRß and GALTRß/RXR complexes in Fig. 1Go), DNA binding, and ligand binding may all influence TR conformation in critical contact regions with coactivators. Recent observations that TR can be phosphorylated further raise the issue of whether additional factors may modulate TR interactions with coactivators (38, 39). Nevertheless, it appears that integration of all these contributions to the conformation of the liganded TR/RXR complex likely is the first critical step that dictates the interactions with corepressors, coactivators, and the basal transcriptional machinery. The summation of these interactions, then, may result in the repression, derepression, and activation of transcription of target genes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Creation of TRß mutants
All expression vectors for TRs were expressed in pcDNA1/Amp (pcDNA) (Invitrogen, San Diego, CA). hTRß, R429Q, G345, TRß-{Delta}N.rTRß, and rTR{alpha} have been previously described (40, 41). R428Q, in pBS (gift from Dr. L. Jameson, Northestern University, Chicago, IL) was subcloned into pcDNA (42). TRAHTM in pcDNA was created using an in vitro mutagenesis kit (Promega, Madison, WI) and a mutagenesis primer that changed codons 223, 224, and 227 from A, H, and T to G, G, and A, respectively (8). TRß-TG and TRß-GT, in which the sequences encoding first zinc and second zinc fingers of human TRß have been swapped with hGR, were generated by PCR amplification of the DNA sequences coding for each zinc finger from TRßnx and GRnx plasmids in pRSV (43) (gifts from Dr. R. M. Evans, Salk Institute, San Diego, CA) and then subcloned into HindIII and KpnI sites of the pcDNA polylinker. TRß-TGT contains the hTRß DBD substituted with the hGR DBD in pRSV (43). AF-2 LBD mutants, 457A-LBD and 457D-LBD, were created by PCR amplification using primers containing mutations of the codon 457 and HindIII restriction site and a primer containing a SmaI restriction site and TRß cDNA sequence starting from codon 174 (encodes an internal methionine). Full-length AF-2 mutants, E457A and E457D, were created by using the same primers containing the mutations of codon 457 and a primer containing a SmaI restriction and TRß cDNA containing the first translational start site methionine. These PCR fragments were isolated, purified, and then subcloned into pcDNA expression vectors. mRXRß in pcDNA has been previously described.

GAL4RXR and GAL4TRß encode amino acids 1–147 of GAL4 DNA-binding sequence and of amino acids 157–410 of the mRXRß (gift of Dr. K. Ozato, NIH, Bethesda, MD) and 173–161 of hTRß-1 LBDs (23, 44).

To generate the UAS-reporter, an oligonucleotide containing the GAL4-binding site, UAS, a previously described 17-bp sequence was used (45). This oligonucleotide contained BamHI and EcoRI restriction sites on either end and was subcloned into the reporter vector, PT109, which contains a viral thymidine kinase minimal promoter and the firefly luciferase cDNA, as previously described (46). Clones were isolated, sequenced, and maxi-prepped by affinity chromatography (Qiagen, Chatsworth, CA) before used in transfections.

Cotransfection Studies
cDNA clones encoding the TRs and TRß mutants in pcDNA expression vector (Fig. 1Go) as well as GAL4, GAL4TRß, and GAL4RXR were used in the cotransfection experiments. Reporter plasmids containing the UAS and the luciferase cDNA in PT109 described above were used (45, 46).

CV-1 cells were grown in DMEM/10% FCS. The serum was stripped of T3 by constant mixing with 5% (wt/vol) AG1-X8 resin (Bio-Rad, Richmond, CA) twice for 12 h at 4 C before ultrafiltration. The cells were transfected with expression (0.1 µg) and reporter (2 µg) plasmids as well as a RSV-TRß-galactosidase control plasmid (1 µg) in 3.5-cm plates using the calcium-phosphate precipitation method (47). Cells were grown for 24 h in the absence or presence of 10-6 M T3 (Sigma, St. Louis, MO) or 9-cis- retinoic acid (Biomol), and harvested. Cell extracts then were analyzed for both luciferase and ß-galactosidase activity to correct for transfection efficiency (48, 49). Except where indicated, the corrected luciferase activities of untreated samples were normalized to the luciferase activities of samples containing pcDNA (vector) and GAL4 expression vectors in the absence of ligand (1-fold basal).


    FOOTNOTES
 
Address requests for reprints to: Paul M. Yen, Molecular and Cellular Endocrinology Branch, NIDDK/NIH, Building 10, Room 8D12, 9000 Rockville Pike, Bethesda, Maryland 20892.

We thank Dr. Ron Evans (Salk Institute, La Jolla, CA), Dr. Larry Jameson (Northwestern University, Chicago, IL), Dr. Keiko Ozato (NIH, Bethesda, MD), and Dr. Samuel Refetoff (University of Chicago, Chicago, IL) for kind provision of plasmids.

Dr. Yen received support from the March of Dimes Foundation for this study.

1 Current address: Molecular and Cellular Endocrinology Branch, NIDDK, NIH, Bethesda, Maryland 02892. Back

Received for publication May 8, 1997. Revision received September 11, 1997. Accepted for publication October 6, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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