Identification of Direct Repeat 4 as a Positive Regulatory Element for the Human TR4 Orphan Receptor
A MODULATOR FOR THE THYROID HORMONE TARGET GENES*

(Received for publication, August 21, 1996, and in revised form, December 5, 1996)

Yi-Fen Lee Dagger §, Huei-Ju Pan Dagger §, J. Peter H. Burbach , Eugene Morkin par and Chawnshang Chang Dagger **

From the Dagger  Comprehensive Cancer Center, University of Wisconsin, Madison, Wisconsin 53792 and Departments of Pathology, Urology and Biochemistry, University of Rochester, Rochester, New York 14642, the  Department of Medical Pharmacology, Utrecht University, 3584 CG Utrecht, The Netherlands, and par  University Heart Center, University of Arizona, Tucson, Arizona  85724

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

While the TR4 orphan receptor (TR4) is able to repress the expression of its target genes via its interaction with the direct repeat 1-hormone response element (DR1-HRE) and DR2-HRE, we now report that TR4 can also induce the transcriptional activity of the reporter gene containing a DR4-HRE via chloramphenicol acetyltransferase assay. Electrophoretic mobility shift assay and Scatchard analysis reveal a strong binding affinity (dissociation constant = 2 nM) between TR4 and DR4-HRE. The induction mediated by TR4 was detected not only in the synthetic DR4-HRE but also in some genes, such as rat alpha -myosin heavy-chain and S14 genes, containing the DR4 or DR4-like motif, which have been suggested to be the response elements for a thyroid hormone receptor. Our data also demonstrate this TR4-mediated gene induction is TR4 dose- and DR4 sequence-dependent. Together, our data suggest that DR4-HRE can be a positive regulatory element for TR4, which may be able to induce the transcriptional activity of the genes containing such positive HREs.


INTRODUCTION

The nuclear receptor superfamily comprises a large group of ligand-dependent transcriptional factors that control the expression of target genes by binding to their cognate hormone response elements (HREs)1 (1-4). It includes the receptors for steroid hormones, thyroid hormones, and retinoids. In addition to the classical receptors, a large number of members in this superfamily have been cloned via their sequence conservation, for which ligands were not found and therefore were classified as orphan receptors (5). The human TR4 orphan receptor (TR4) was initially isolated by our laboratory from human prostate and testis cDNA libraries by degenerative polymerase chain reaction cloning (6). TR4 shows a high degree of nucleotide sequence homology with the TR2-11 orphan receptor and thus forms a unique subfamily in this superfamily (7). Recently, TR4 has also been identified from human lymphoblastoma cells, called TAK1 (8). Northern blot analysis shows that TR4 is expressed in many tissues, being predominantly located in granule cells of the hippocampus and the cerebellum (6). This implies that the TR4 may regulate the specific gene function in a cell-specific manner.

A cysteine-rich cluster that comprises two zinc finger-like structures can be easily identified within the DNA binding domain of the nuclear receptors (9-12). Three amino acids in the stem of the first zinc finger (the P box) are critical to specify the half-site sequence, while 5 amino acids in the second finger (the D box) can alter the selection pattern of the half-site spacing. On the basis of its distinct P box, TR4 can be classified into the estrogen receptor/thyroid hormone receptor subfamily which displays cross-recognition of the palindromic or direct repeat (DR) HREs. These HREs consist of a core half-site, AGGTCA, or related sequence (13-15). Our previous data suggested that TR4 preferred to bind to the HREs, which consist of two DRs of consensus half-site, rather than a palindrome.2 Furthermore, TR4 can repress the retinoic acid-induced transactivation by competitive DNA binding to the response elements of other receptors (i.e. DR5 for retinoic acid receptor (RAR) and DR1 for retinoid X receptor (RXR)).2 In addition, the TR4-mediated suppression can also be found in the gene promoter containing DR2-like response element that is located in the +55 region of the SV40 major late promoter (16).

All of the above findings suggest that TR4 may function as a repressor to block other receptor-mediated transcriptional activities through competitive DNA binding to the same HREs. In the present study, we tested the regulation of TR4 on a DR4-HRE which has been shown to be a hormone response element for thyroid hormone receptor (T3RE) by using a reporter gene assay (17-19). Through these studies, we found that the TR4 can induce gene expression through the interaction with DR4-HRE in a sequence- and dose-dependent manner. In addition to repressing its target genes, our data provide the first evidence to show that TR4 can also activate its target genes, through a DR4- or DR4-like T3RE. This finding may expand our understanding of the physiological function of the TR4 orphan receptor.


MATERIALS AND METHODS

Plasmid Construction

For the transient transfection or coupled in vitro transcription and translation of the full-length TR4 protein, the pCMX-TR4 and pET14b-TR4 plasmids were constructed as described, respectively (16). pCD-TRalpha 1, the expression vector for thyroid hormone receptor alpha , was provided by Dr. Leslie J. Degroot. The reporter plasmids -32TK-CAT and TK-(DR4)2-CAT, either without or with two copies of DR4 (5'-GATATAGGGGTCAAATAAGGTCAAATG-3', Fig. 1A), were kindly provided by Dr. RC. Ralff. TK-(mDR4)-CAT containing one copy of the mutated DR4 (5'-GATCTAGAATAAATG-3') inserted into the BamHI site of -32TK-CAT plasmid was generated as a negative control. The reporter genes used pCATp reporter genes fused with either the 5'-flanking region of rat alpha -myosin heavy chain (alpha -MHC) (from -374 to +420) (17), the far upstream regulatory region (FUR) (from -2952 to -2448) in the rat S14 gene (FUR1/2/3-CAT) (18), or the long terminal repeat of the human immunodeficiency virus I (from -454 to +82, HIVLAILTR.CAT) (19), which contains the T3RE motif in the natural promoter region.


Fig. 1. Binding of in vitro expressed TR4 to DR4. A, the sequences of the wild type DR4 and mutated DR4 oligonucleotides. B, 32P-labeled DR4 oligomers were used in EMSA with 1 µl of in vitro translated TR4. A 100-fold excess unlabeled wild type DR4 (wDR4) (lane 3) or mutant DR4 (mDR4) (lane 4) was added as a competitor. 1 µl of anti-TR4 monoclonal antibody (G232-303.4) was also added (lane 6) and anti-androgen receptor monoclonal antibody (N1-15) was used as a control (lane 5). The migration position of the specific binding formed by the DNA-protein complex and the supershifted band formed by adding monoclonal antibody are indicated as an arrowhead or arrow, respectively.
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Coupled in Vitro Transcription and Translation

Expression plasmids pET14b-TR4 and pCD-TRalpha 1 were in vitro transcribed and translated by the TNT system (Promega) as described previously (16).

Electrophoretic Mobility Shift Assay (EMSA)

EMSA analysis was performed according to the methods described by Cooney et al. (20). In general, the double-stranded DR4 oligonucleotides were end-labeled with a [gamma -32P]ATP (specific activity = 6000 Ci/mmol, DuPont NEN) by T4 polynucleotide kinase to 2-8 × 108 cpm/µg. For competitive DNA binding reactions, 100-fold excess of cold double-stranded DR4 or mDR4 oligonucleotides were mixed with the proteins prior to the addition of labeling probe to the reactions. For antibody supershift analysis, 1 µl of the monoclonal anti-TR4 antibody (G232-303.4) was added to the reaction. As a control for supershift assays, the monoclonal anti-androgen receptor antibody (N1-15) (21) was added. Gels were fixed in 50% ethanol and 10% acetic acid for 30 min before drying. The radioactive gels were analyzed by either PhosphorImager (Molecular Dynamics Inc.) or autoradiography.

Scatchard Analysis

The DNA-protein binding assay was performed as described previously (16). In brief, free probes (32P-DR4) and DNA-protein complexes were resolved in native gel and quantified by scintillation counting. The dissociation constant (Kd) was determined from the minus reciprocal of the slope of the line generated from the empirical data.

Transient Tansfection

HepG2 cells were routinely maintained in Dulbecco's modified Eagle's medium with 5% heat-inactivated fetal bovine serum. HepG2 cells (3 × 105) were seeded in 6-cm culture dishes 24 h before transfection. The medium was changed to Dulbecco's modified Eagle's medium with 5% charcoal dextran-treated serum at least 1 h before transfection. The cells were transfected using a modified calcium phosphate precipitation method (22). To normalize the transfection efficiency, the beta -galactosidase expression vector was co-transfected.

Statistics

The data are presented as mean ± S.D. One-way analysis of variance was used to determine the differences among multiple groups. Student's t test was used for analyzing differences between two groups. The degree of significance (p value) was shown with asterisks. p < 0.05 was accepted as the level of statistical significance.


RESULTS

TR4 Specifically Binds to DR4, the AGGTCA Motif with 4-Nucleotide Spacing

EMSA was used to determine binding specificity for TR4 to DR4-HRE. In vitro translated TR4 protein was incubated with 32P-labeled DR4-HRE and analyzed on a 5% polyacrylamide gel. As shown in Fig. 1B, a specific DNA-protein complex (indicated with arrowhead, lane 2) was identified that has a migration distinct from the nonspecific DNA-protein complexes in the presence of mock-translated product (lane 1). The binding specificity was further confirmed by adding excess unlabeled DR4-HRE or mutated DR4 (mutated DR4-HRE, in which the third G was replaced with C) (Fig. 1A). Competition by 100-fold excess unlabeled DR4-HRE abolished the specific DNA protein complex (lane 3). In contrast, this complex could not be abolished completely with the unlabeled mutant DR4-HRE (lane 4). This DNA-protein complex could not be affected when androgen receptor monoclonal antibody (N1-15) was added (lane 5). In addition, when the anti-TR4 monoclonal antibody (G232-303.4) was added in the reaction, a supershifted band made by DNA-receptor-antibody complex was visible (lane 6).

All these data clearly demonstrated that the TR4 can bind specifically to DR4-HRE. We also calculated the binding affinity between TR4 and DR4-HRE by the Scatchard analysis. Fixed amounts of in vitro translated TR4 proteins were incubated with an increasing amount of 32P-labeled DR4 (0.1-12.8 ng) and resolved in EMSA. As shown in Fig. 2, Scatchard analysis displays a single binding component with a dissociation constant (Kd) of 2 nM and Bmax of 0.075 nM. These data fit the Kd range for steroid receptors and their HREs.


Fig. 2. Scatchard analysis of TR4 to DR4. 1 µl of in vitro translated TR4 protein was incubated with serially diluted 32P-labeled DR4 (concentrations from 0.1 to 12.8 ng), and resolved in EMSA. After autoradiography, the respective bands were excised, placed in scintillation fluid, and quantified directly in a scintillation counter. The ratio activity between specific DNA-protein binding (bound, nM) and free DNA probe with respect to specific DNA-protein binding (bound/free) was plotted. The dissociation constant (Kd) and Bmax values were generated from the Edba program (Biosoft).
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TR4 Mediates Dose-dependent TK-(DR4)2-CAT Transcriptional Activation

As shown above, TR4 can bind to a DR4-HRE with high specificity and affinity. We were then interested in knowing the consequences of this binding. The reporter gene TK-(DR4)2-CAT and the TR4 expression (pCMX-TR4) plasmid were co-transfected into HepG2 cells. Surprisingly, we found that TR4 itself was able to activate CAT activity (Fig. 3A, lane 3). This transactivation could not be observed when pCMX-TR4 was co-transfected with the reporter plasmids without DR4-HRE (-32TK-CAT, lane 1), or with the mutated DR4-HRE (TK-(mDR4)-CAT, lane 2). Moreover, the CAT activity was increased as increasing amounts (from 0.01 to 1 µg) of pCMX-TR4 were co-transfected (Fig. 3B, lanes 1-6) Transcriptional activity reached a plateau when 2 µg of pCMX-TR4 was co-transfected (lane 7). In summary, these data clearly demonstrated that TR4 induced transcriptional activation is TR4 dose- and DR4-HRE sequence-dependent.


Fig. 3. T3-independent activation of TK-(DR4)2-CAT by TR4. A, -32TK-CAT, TK-(mDR4)-CAT, or TK-(DR4)2-CAT reporter plasmids (3 µg of each) were transfected into HepG2 cells with or without co-transfection of TR4 expression vector (pCMX-TR4, 1 µg). At the 3rd day after transfection, cells were harvested for determination of CAT activities. The relative CAT conversion was determined by comparing the CAT activity in each treatment with the one without co-transfection of 1 µg of pCMX-TR4. Significant differences were determined by Student's t test and marked an asterisk. *p < 0.001 compared with pSG5-transfected groups. B, dose response of TK-(DR4)2-CAT activation by TR4. HepG2 cells were transfected with TK-(DR4)2-CAT reporter plasmids (3 µg of each) and co-transfected with indicated increasing amounts of pCMX-TR4 (from 0 to 2 µg). The percentage of CAT conversion was plotted. All CAT assays were normalized for the level of beta -galactosidase activity. Significant differences (p < 0.005) of CAT activity induced by TR4 at different doses were determined by one-way analysis of variance. Each value represents the mean ± S.D. of four independent experiments.
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The Correlation between TR4 and TRalpha 1 in the Transactivation of TK-(DR4)2-CAT

All of the data above suggest that DR4-HRE is a positive novel response element for the TR4. Previous reports suggest that the DR4-HRE can also serve as the response element for the T3RE. Therefore we investigated how these two receptors interacted with each other on the same target sequence by designing the following experiments. First, a fixed amount (2 µg) of thyroid hormone receptor expression vector, pCD-TRalpha 1, was transfected singly or co-transfected with increasing amounts of pCMX-TR4 (from 0.125 to 1 µg) in the absence of T3. As shown in Fig. 4A, CAT activities were activated only when pCMX-TR4 was transfected (lane 1 versus 2). No activation was detected when pCD-TRalpha 1 was transfected alone (lane 1 versus 3). The induction was significant increasing (p < 0.005) by increasing amounts of pCMX-TR4 (lanes 3-7). The induced CAT activities mediated by TR4 were slightly reduced when the unliganded pCD-TRalpha 1 (2 µg) was co-transfected (lane 2 versus 7). However, the reduction of TR4-mediated CAT activation by unliganded TRalpha 1 was not very significant (p < 0.1) by Student's t test. This phenomenon was also observed by co-transfection of pCMX-TR4 (1 µg) with increasing amounts of unliganded pCD-TRalpha 1 (Fig. 4C, lanes 3-7). The activities induced by TR4 (1 µg) was reduced 50% when 2.0 µg of unliganded TRalpha 1 was co-transfected. (Fig. 4C, lane 3 versus 7). Again, the dose of unliganded TRalpha 1 does not significantly (p < 0.1) repress the activation of CAT activities by TR4. This may be due to the fact that TR4 is more potent than liganded TRalpha 1 in the activation of TK-(DR4)2-CAT reporter gene. The tendency for repression of TR4-mediated transcriptional activity by the unliganded TRalpha 1 is consistent with previous reports (23-25) in which suggested that unliganded TRalpha 1 may act as a gene silencer.


Fig. 4. The combination effect of TR4 and thyroid hormone receptor (TRalpha 1) on TK-(DR4)2-CAT. TK-(DR4)2-CAT (3 µg) was co-transfected into HepG2 cells with pSG5, pCMX-TR4, or pCD-TRalpha 1. In panels A and B, fixed amounts of TRalpha 1 (2 µg) were co-transfected with indicated increasing amounts of pCMX-TR4 (from 0 to 1 µg). In panels C and D, fixed amounts of pCMX-TR4 (1 µg) and increasing amounts of pCD-TRalpha 1 (from 0 to 2 µg) were co-transfected as indicated. Twenty-four hours after transfection, the cells were left untreated (panels A and C) or treated (panels B and D) with 10-7 M T3 and harvested for determination of CAT activities. All CAT assays were normalized for the level of beta -galactosidase activity. In panel A, the dose of TR4 significantly (*p < 0.005) affects the induction of DR4-CAT reporter gene activity in the presence of unliganded TRalpha 1. In panel C, the dose of unliganded TRalpha 1 does not significantly (p < 0.1) repress the induction of DR4-CAT activity by 1 µg of TR4. Each value represents the mean ± S.D. of three independent experiments. The induction fold was compared with the transfection of TK-(DR4)2-CAT plus pSG5. Significant differences of CAT activity affected by TR4 or TRalpha 1 at different doses were determined by one-way analysis of variance.
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In contrast, in the presence of T3 (10-7 M), both pCD-TRalpha 1 and pCMX-TR4 can activate CAT activities up to 15- and 22-fold alone, respectively (Fig. 4, B and D, lanes 1-3). The transcriptional activity slightly increased when we co-transfected 2 µg of pCD-TRalpha 1 and increasing amounts of pCMX-TR4 (0.125 to 1 µg) (Fig. 4B, lanes 3-7). Furthermore, when a fixed amount of pCMX-TR4 (1 µg) was transfected together with increasing amounts of pCD-TRalpha 1 (from 0.5 to 2 µg) (Fig. 4D, lanes 3-7), the results showed that CAT activity induced by TR4 did not increase significantly by adding additional TRalpha 1. This may suggest that transcriptional activity could be saturated by expression of 1 µg of TR4 alone.

TR4 Activates the Transcriptional Activities of Some Genes Containing T3RE Motif

As described above, TR4 can recognize synthetic DR4-HRE and activate TK-(DR4)2-CAT reporter gene in a dose- and DNA sequence-dependent manners. We then explored whether this TR4-mediated CAT induction could also be applied to the DR4-T3RE-containing or nonclassical T3RE natural promoters. The sequences corresponding to T3RE were shown in Fig. 5A with an arrow or underline. The first gene used was from the 5'-flanking region of rat alpha -MHC gene (from -347 to +420), which contains a DR4-HRE that serves as a T3RE (17). The expected results were shown in Fig. 5B, panel a. The transcriptional activity of this gene can be induced up to 9.5-fold with co-transfection of pCMX-TR4. The second gene tested was the FUR of the rat S14 gene (from -2952 to -2448) which contains multiple T3REs that synergize with each other in their responsiveness to T3 (18, 26). As shown in Fig. 5B, panel b, the transcriptional activity of rS14 can be induced up to 6.3-fold upon co-transfection with pCMX-TR4. The third native gene we tested was the HIV-LTR (from -454 to +82), which contains a nonclassical T3RE, GGCGGG, and can be activated by unliganded TRalpha 1 (27). A 3.9-fold induction mediated by pCMX-TR4 can also be observed (Fig. 5B, panel c). The differences of CAT activity obtained between TR4-transfected and control in these three native T3RE responsive genes were considered significant (p < 0.05) based by Student's t test. All these data suggest that the DR4-T3RE as well as some nonclassical T3REs can serve as potential target sequences for both T3R and TR4.


Fig. 5. Effect of TR4 on T3RE-containing natural promoter genes. A, the sequence for three natural genes which contain potential T3REs. The sequences for the potential T3RE are indicated with arrows or underlined. B, three CAT reporter constructs (3 µg of each) were co-transfected with pCMX-TR4 (1 µg) or pSG5. Three reporter genes were used as following: panel a represents the reporter gene fused with the 5'-flanking region of rat alpha -MHC (from -374 to +420), which contains DR4-T3RE; panel b represents the pCATp fused with rat S14 promoter (FUR1/2/3: from -2952 to -2448), which contains multiple T3REs; panel c represents the pCATp fused with HIV-LTR (from -454 to +82), which contains nonclassical T3RE. The fold induction was calculated by the comparison of CAT activity of transfection of pSG5 with pCMX-TR4. Significant differences were determined by Student's t test and marked with asterisks (*p < 0.005 compared with pSG5-transfected groups). All CAT assays were normalized for the level of beta -galactosidase activity. Each value represents the mean ± S.D. of four independent experiments.
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To further define the correlation between TR4 and TRalpha 1 in these natural promoter constructions, we then tested the overexpression of both pCMX-TR4 and pCD-TRalpha 1 in the reporter gene assay. Fig. 6A showed that CAT activities were significantly (p < 0.05) induced by the transfection of pCMX-TR4 alone in all reporter plasmids we had tested, but induction could not be observed in the transfection of unliganded pCD-TRalpha 1. In the absence of T3, the activation induced by TR4 was slightly reduced by unliganded TRalpha 1 in synthetic DR4, alpha -MHC, and S14 reporter gene constructs but not in HIV-LTR reporter gene. These results agree with a previous report in which HIV-LTR was shown to be activated by unliganded TRalpha 1, and the addition of T3 could reverse this effect (27). On the other hand, in the presence of T3 (Fig. 6B), cotransfection of pCMX-TR4 with pCD-TRalpha 1 slightly increased the activation as compared with the single receptor transfection but the increasing effect is not very significant. Together, our data provides the first evidence showing that the TR4 is capable of activation of CAT reporter genes containing a DR4-T3RE. This is true for both synthetic and native T3REs as well as other nonclassical T3REs.


Fig. 6. The correlation between TR4 and TRalpha 1 in three native T3RE containing reporter genes. pCMX-TR4, pCD-TRalpha 1 or pSG5 were co-transfected with TK-(DR4)2-CAT and three natural T3RE-containing reporter genes (alpha -MHC, rS14, and HIV-LTR). Twenty-four hours after transfection, cells were left either untreated (panel A) or treated (panel B) with 10-7 M T3. Significant differences (p < 0.005) between pSG5-transfected with TR4-transfected or liganded TRalpha 1-transfected were determined by Student's t test and marked with asterisks. All CAT assays were normalized for the level of beta -galactosidase activity. Each value represents the mean ± S.D. of four independent experiments.
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DISCUSSION

Like the TR2 orphan receptor, TR4 can also serve as a repressor in RAR/RXR-mediated gene induction and transcription from the major late promoter of the SV40+55 gene (16, 22, 28). In the present study, we showed TR4 can also induced the transcriptional activity of genes containing a DR4 or DR4-like sequence. These data suggest that the selectivity of the receptor response is dependent on the core motif, including the spacing, orientation, and precise sequence composition of the adjacent core motifs. In Fig. 1, when we replaced the third G with C in DR4-HRE (mDR4), an excessive amount of unlabeled mDR4 could not abolish with the specific DNA-receptor complex completely. This suggests that the precise sequence of the core motif plays an essential role in TR4-mediated gene regulation, a result in agreement with our previous studies as the TR2 orphan receptor (22, 28). In addition, the spacing of core binding motifs may also be a key factor in dictating specificity of transcriptional response to a transcriptional factor. TR4 can either induce or suppress transcription through the binding to HREs with same sequence of the core binding motif but different spacing. Activation or repression may be dependent on different spacing of the motifs (DR1/DR5 for the repression of RAR/RXR-mediated gene induction and DR2 in suppression of SV40 gene versus DR4 in induction of alpha -MHC and S14 genes). Similar results are also observed in the case of the thyroid responsive pathway, as transactivation was observed only on the unspaced palindromic element (PAL), a native rat growth hormone T3RE but not on the 3-base pair-spaced vitellogenin A2 estrogen response element, PAL(+3) (29). Interestingly, when we tested the TR4-mediated induction on different native T3REs, we found that the levels of induction are different among these three T3REs. The order for the TR4-mediated induction among these three native gene promoters is alpha -MHC > rS14 > HIV-LTR. This implies that TR4 had a better induced effect on the DR4-T3REs than TRalpha 1 on such nonclassical T3REs. This suggests that the orientation, and the sequence composition of these adjacent putative core motifs are also very important in the determination of gene regulation. Overall, the combination of core motif spacing, orientation the sequence composition of the core motif and adjacent area could determine the pattern of responsiveness exhibited by genes that are regulated by TR4.

However, the molecular structure of the receptor also contributes to the specificity of gene regulation. For example, the two helical regions in the DNA binding domain of the receptor may also contribute to the specificity of base-specific contacts (30, 31). The third helix formed between the two helices can also serve as a gap to separate two core binding motif (32, 33). When the DNA binding domain of the receptor binds to HREs, the receptor may change its conformation and allow access to different factor(s). As a result, TR4 may bind to different co-factors and contribute to both positive or negative gene regulation through different protein-protein interactions (34). Previous reports have suggested that other nuclear receptors, such as RXR, can serve as a common heterodimerization partner for several nuclear receptors, including the thyroid hormone receptors and retinoic acid receptors (35). The intrinsic binding properties of RXR are masked in such T3R-RXR and RAR-RXR heterodimers. In contrast, RXR is active as a non-DNA-binding co-factor with the NGFI-B/nurr1 orphan receptors (35). Although we were unable to demonstrate the interaction between RXR and TR4, it is still possible that TR4 may need other co-factors for proper function. Using a yeast-two hybrid system, we are in the process of isolating several TR4-associated proteins.3 Recently, COUP-TF has been shown to inhibit the transcriptional activities of T3R, 1,25-dihydroxyvitamin D3 receptor (36), RAR, RXR (37), the peroxisome proliferator-activated receptor (38, 39), and hepatocyte nuclear factor 4 (40). The molecular mechanism of COUP-TF-mediated transcriptional repression may also be involved in some other activator(s) (41). Together, these findings suggest that interactions among proteins may contribute to receptor-mediated gene induction or suppression.

Ligands have always played a central role in the activation of steroid hormone receptors. For example, unliganded T3R is associated with cellular co-repressor p270/N-CoR which serves as a gene silencer (24). Upon ligand binding, T3R dissociates with the co-repressor and actively turns on its target genes (23, 42). However, a similar effect was also observed in this study. Unliganded TRalpha 1 has a potential suppressive effect on the TR4-mediated transactivation in both synthetic DR4 or native T3RE-CAT reporter genes, although the effect is not very significant (p < 0.1). One of the potential explanation is that TR4 has higher binding affinity than TRalpha 1 to T3REs. Therefore, TR4 may compete stronger than TRalpha 1 on binding to the same HRE to activate its target gene. On the other hand, the physiological function of orphan receptors still remains obscure because information regarding ligand is lacking. Nevertheless, the induced CAT activity shown in this study may still provide us a useful assay system for searching for potential ligands or activators for the TR4 orphan receptor.

What then is the relationship between TR4 and TRalpha 1, since they both recognize the same HRE? Transient transfection studies show that TR4 can activate CAT activity in the presence or absence of T3 suggesting that T3 is not the ligand or activator for TR4. In contrast, T3R can only activate transcriptional activity when the ligand, T3, is present. These results suggest that two different receptors may use different ligands to induce similar or different sets of target genes. Another possibility is that although two different receptors can recognize the same HRE, they may function differently and independently in gene regulation. For example, T3R can also bind with high affinity to estrogen response element and serves to antagonize estrogen-dependent transcriptional activation (15). In our present study, we found that the T3-induced transactivation of TRalpha 1 could be overwhelmed by TR4. In addition, the repression effects from unliganded TRalpha 1 could be overcome by adding more TR4. The dominant effect of TR4 may be due to its higher binding affinity to DR4-HRE as compared with that of TRalpha 1 (data not shown). Together, our data suggested TR4 may play an important role in TRalpha -mediated gene regulation.

In summary, our data suggest that the ability of TR4 to induce genes with T3RE-DR4-HRE may provide us with a model system for future studies in determining the mechanisms to determine how the TR4 activates and represses its target genes. Moreover, this TR4-mediated induction system may provide a potential assay for finding a possible ligand and/or activators for TR4.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA55639 and DK47258.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   The first two authors contributed equally to this paper.
**   To whom correspondence should be addressed: Departments of Pathology and Biochemistry, University of Rochester, 601 Elmwood Ave., Box 626, Rochester, NY 14642. Tel.: 716-275-3184; Fax: 716-273-1027.
1   The abbreviations used are: HRE, hormone response element; TR4, TR4 orphan receptor; DR, direct repeat; alpha -MHC, alpha -myosin heavy chain; FUR, far upstream regulatory region; HIV, long terminal repeat of the human immunodeficiency virus I; T3R, thyroid hormone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; RA, retinoic acid; EMSA, electrophoretic mobility shift assay; CAT, chloramphenicol acetyltransferase.
2   Y.-F. Lee and C. Chang, manuscript in preparation.
3   S. Yeh and C. Chang, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Dr. Leslie J. Degroot for thyroid hormone receptor expression vector, pCD-TRalpha 1. We also thank Dr. H. C. Towle for kindly providing the rat S14 gene promoter, Dr. J. A. A. Ladias for HIV-LTR reporter gene, and Dr. Alan Saltzman for his helpful comments and editing on the manuscript.


REFERENCES

  1. Evans, R. M. (1988) Science 240, 889-895 [Medline] [Order article via Infotrieve]
  2. Beato, M. (1989) Cell 56, 335-344 [Medline] [Order article via Infotrieve]
  3. Amero, S. A., Matunis, M. J., Matunis, E. L., Hockensmith, J. W., Raychaudhuri, G., and Beyer, A. L. (1993) Mol. Cell. Biol. 13, 5323-5330 [Abstract]
  4. Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486 [CrossRef][Medline] [Order article via Infotrieve]
  5. O'Malley, B. W., and Conneely, O. M. (1992) Mol. Endocrinol. 6, 1359-1361 [Medline] [Order article via Infotrieve]
  6. Chang, C., Lopa da Silva, S., Ideta, R., Lee, Y., Yeh, S., and Burbach, J. P. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6040-6044 [Abstract]
  7. Chang, C., Kokontis, J., Acakpo-Satchivi, L., Liao, S., Takeda, H., and Chang, Y. (1989) Biochem. Biophys. Res. Commun. 165, 735-741 [Medline] [Order article via Infotrieve]
  8. Hirose, T., Fujimoto, W., Tamaai, T., Kim, K. H., Matsuura, H., and Jetten, A. M. (1994) Mol. Endocrinol. 8, 1667-1680 [Abstract]
  9. Green, S., and Chambon, P. (1987) Nature 325, 75-78 [CrossRef][Medline] [Order article via Infotrieve]
  10. Hollenberg, S. M., and Evans, R. M. (1988) Cell 55, 899-906 [Medline] [Order article via Infotrieve]
  11. Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., and Yamamoto, K. R. (1988) Nature 334, 543-546 [CrossRef][Medline] [Order article via Infotrieve]
  12. Archer, T. K., Hager, G. L., and Omichinski, J. G. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7560-7564 [Abstract]
  13. Glass, C. K., Holloway, J. M., Devary, O. V., and Rosenfeld, M. G. (1988) Cell 54, 313-323 [Medline] [Order article via Infotrieve]
  14. Brent, G. A., Harney, J. W., Chen, Y., Warne, R. L., Moore, D. D., and Larsen, P. R. (1989) Mol. Endocrinol. 3, 1996-2004 [Abstract]
  15. de Verneuil, H., and Metzger, D. (1990) Nucleic Acids Res. 18, 4489-4497 [Abstract]
  16. Lee, H.-J., Lee, Y., Burbach, J. P. H., and Chang, C. (1995) J. Biol. Chem. 270, 30129-30133 [Abstract/Free Full Text]
  17. Tsika, R. W., Bahl, J. J., Leinwand, L. A., and Morkin, E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 397-383
  18. Liu, H. C., and Towle, H. C. (1994) Mol. Endocrinol. 8, 1021-1037 [Abstract]
  19. Ladias, J. A. A. (1994) J. Biol. Chem. 269, 5944-5951 [Abstract/Free Full Text]
  20. Cooney, A. J., Tsai, S. Y., O'Malley, B. W., and Tsai, M. J. (1992) Mol. Cell. Biol. 12, 4153-4163 [Abstract]
  21. Chang, C., Whelan, C. T., Popovich, T. C., Kokontis, J., and Liao, S. (1989) Endocrinology 123, 1097-1099
  22. Lee, H.-J., and Chang, C. (1995) J. Biol. Chem. 270, 5434-5440 [Abstract/Free Full Text]
  23. Casanova, J., Helmer, E., Selmi-Ruby, S., Qi, J. S., Au-Fliegner, M., Desai-Yajnik, V., Koudinova, N., Yarm, F., Raaka, B. M., and Samuels, H. H. (1994) Mol. Cell. Biol. 14, 5756-5765 [Abstract]
  24. Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., and Glass, C. K. (1995) Nature 377, 397-404 [CrossRef][Medline] [Order article via Infotrieve]
  25. Qi, J. S., Desai-Yajnik, V., Greene, M. E., Raaka, B. M., and Samuels, H. H. (1995) Mol. Cell. Biol. 15, 1817-1825 [Abstract]
  26. Zilz, N. D., Murray, M. B., and Towle, H. C. (1990) J. Biol. Chem. 265, 8136-8143 [Abstract/Free Full Text]
  27. Rahman, A., Esmaili, A., and Saatcioglu, F. (1995) J. Biol. Chem. 270, 31059-31064 [Abstract/Free Full Text]
  28. Lin, T.-M., Young, W.-J., and Chang, C. (1995) J. Biol. Chem. 270, 30121-30128 [Abstract/Free Full Text]
  29. Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) Cell 65, 1255-1266 [Medline] [Order article via Infotrieve]
  30. Luisi, B. F., Xu, W. X., Otwinowski, Z., Freedman, L. P., Yamamoto, K. R., and Sigler, P. B. (1991) Nature 352, 497-505 [CrossRef][Medline] [Order article via Infotrieve]
  31. Schwabe, J. W., Chapman, L., Finch, J. T., and Rhodes, D. (1993) Cell 75, 567-578 [Medline] [Order article via Infotrieve]
  32. Wilson, T. E., Paulsen, R. E., Padgett, K. A., and Milbrandt, J. (1992) Science 256, 107-110 [Medline] [Order article via Infotrieve]
  33. Kurokawa, R., Yu, V. C., Naar, A., Kyakumoto, S., Han, Z., Silverman, S., Rosenfeld, M. G., and Glass, C. K. (1993) Genes Dev. 7, 1423-1435 [Abstract]
  34. Glass, C. K. (1994) Endocr. Rev. 15, 391-407 [Medline] [Order article via Infotrieve]
  35. Forman, B. M., Umesono, K., Chen, J., and Evans, R. M. (1995) Cell 81, 541-550 [Medline] [Order article via Infotrieve]
  36. Cooney, A. J., Leng, X., Tsai, S. Y., O'Malley, B. W., and Tsai, M. J. (1993) J. Biol. Chem. 268, 4152-4160 [Abstract/Free Full Text]
  37. Tran, P., Zhang, X. K., Salbert, G., Hermann, T., Lehmann, J. M., and Pfahl, M. (1992) Mol. Cell. Biol. 12, 4666-4676 [Abstract]
  38. Miyata, K. S., Zhang, B., Marcus, S. L., Capone, J. P., and Rachubinski, R. A. (1993) J. Biol. Chem. 268, 19169-19172 [Abstract/Free Full Text]
  39. Baes, M., Castelein, H., Desmet, L., and Declercq, P. E. (1995) Biochem. Biophys. Res. Commun. 215, 338-345 [CrossRef][Medline] [Order article via Infotrieve]
  40. Kimura, A., Nishiyori, A., Murakami, T., Tsukamoto, T., Hata, S., Osumi, T., Okamura, R., Mori, M., and Takiguchi, M. (1993) J. Biol. Chem. 268, 11125-11133 [Abstract/Free Full Text]
  41. Leng, X., Cooney, A. J., Tsai, S. Y., and Tasi, M.-J. (1996) Mol. Cell. Biol. 16, 2332-2340 [Abstract]
  42. Thompson, C. C., and Evans, R. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3494-3498 [Abstract]

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