©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ligand-independent and -dependent Functions of Thyroid Hormone Receptor Isoforms Depend upon Their Distinct Amino Termini (*)

Anthony N. Hollenberg (§) , Tsuyoshi Monden , Fredric E. Wondisford

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Isoform specificity likely plays a large role in the ability of the thyroid hormone receptor (TR) to specifically regulate gene expression in both the presence and absence of its cognate ligand, triiodothyronine. To investigate further the mechanism of isoform specificity of human TRs (TR1 and TR1), we have examined their functional effects on positive thyroid hormone response elements (TREs) both in the presence and absence of ligand. TR1 was greater than 2-fold more potent than TR1 on both TREs studied, in terms of both ligand-independent repression and ligand-dependent stimulation. By creating a number of chimeric and mutant receptors, we have established that the increased functional potency of TR1 is due to its unique amino terminus. Deletion or substitution of the TR1 amino terminus leads to a loss of both its ligand-independent and -dependent functions on positive TREs. Furthermore, the TR1 amino terminus antagonizes homodimer formation on the positive TREs studied. TR constructs, which contain the TR1 amino terminus, are unable to form homodimers and form exclusively heterodimers with RXR on direct repeat and palindromic TREs. Deletion of the amino terminus from either TR isoform leads to preferential homodimer formation, which suggests that the TR amino terminus is important for relative heterodimerization capability. From these data, we conclude that TR1 isoform specificity on positive TREs resides predominantly in its amino terminus through its ability to favor heterodimerization with the retinoid X receptor or other nuclear proteins.


INTRODUCTION

Thyroid hormone regulates target gene expression through cis-acting response elements or TREs,()which bind the thyroid hormone receptor (TR) and auxiliary proteins such as the retinoid X receptor (RXR)(1, 2, 3) . Several investigators have shown that TREs present in positively regulated elements are repeats of a half-site consensus motif (AGGTCA) oriented as a direct repeat with a 4 base pair gap (DR+4), a palindrome (PAL), or an inverted palindrome(4, 5, 6) . The TR interacts with these response elements as a monomer, homodimer, or heterodimer with RXR isoforms. Heterodimerization with RXR greatly enhances positive induction of gene expression by thyroid hormone (7-10). Besides its ligand-dependent properties, the TR functions as a ligand-independent silencer on positive TREs(11, 12) and as ligand-independent activator on negative TREs (13, 14) in mammalian cells. These ligand-independent properties appear to be dependent on TRRXR heterodimer, as TR receptor mutants that cannot form homodimers still possess potent ligand-independent function(13) .

Further complexity in thyroid hormone signaling is added by the presence of multiple isoforms of the TR(15) . The two major isoforms, TR1 and TR1, are encoded by separate genes but are relatively homologous except for distinct amino termini. Each of these receptors has alternatively spliced variants TR2 and TR2, respectively, which may be expressed in a tissue-specific fashion (16) to regulate gene expression. Isoform specificity has been demonstrated on a unique negative TRE present in the Rous sarcoma virus promoter where the unique amino terminus of TR1 allows for ligand-independent activation(17) . The TR domain responsible for isoform-specific effects on positive TREs has not been demonstrated, although TR1 is more potent as a ligand-dependent activator on positive TREs (18, 19) and appears to be relatively defective in its ability to homodimerize on positive TREs(20, 21) . Furthermore, differential expression of TR isoforms has been observed during development(22) , suggesting that TR isoform specificity plays a role in differential gene expression. In the following report, we demonstrate that isoform specificity on positive TREs is a function of both ligand-independent and -dependent properties of the TR and that these enhanced properties map to the unique amino terminus of TR1. In addition, the amino terminus of TR1 inhibits homodimerization on positive TREs favoring the formation TRRXR heterodimers. These distinct properties of the amino terminus of TR1 allow for TR isoform-specific regulation of target gene expression.


MATERIALS AND METHODS

Plasmid Construction

The positive TREs consist each of two copies of PAL (a gift of J. L. Jameson, Northwestern University, Chicago) and DR+4 inserted upstream of the TK109 promoter in the luciferase vector pALUC(23, 24) , which contains a trimerized SV40 poly(A) termination site that prevents transcriptional read through.

The cDNAs encoding human TR1, TR1, and chimeric and mutated TRs were inserted into the expression vector pKCR-2, which employs the SV40 early promoter(25) . Chimeric TRs were constructed using SacI and PstI restriction sites present in the TRs. A SacI site was introduced into TR1 at amino acid 52 to allow for exchanges of the amino termini of the different TR isoforms. Amino-terminal deletions were constructed using the polymerase chain reaction to introduce a Kozak initiation sequence at the beginning of the DNA binding domain of each isoform. The integrity of all constructs was confirmed by restriction endonuclease digestion and dideoxy sequencing.

Cell Culture and Transient Transfection

The CV-1 cell line was maintained in Dulbecco's modified Eagle's medium supplemented with L-glutamine, 10% fetal calf serum, 100 µg/ml penicillin, 0.25 µg/ml streptomycin, and amphotericin subsequently referred to as culture medium. Transient transfections were performed in six well plates on subconfluent cells. 1.6 µg of reporter construct with 80 ng of receptor expression vector and 80 ng of pKCR-2 vector alone (except where noted) were introduced into each well using the calcium phosphate technique without glycerol shock. 12-15 h after transfection, the cells were washed with phosphate-buffered saline, and the culture medium was replaced with culture medium with fetal bovine serum treated with anion exchange resin and charcoal (type AGX-8, analytical grade (Bio-Rad), and activated charcoal (Sigma)). 24 h after transfection, 10 nM T was added. 42-48 h after transfection, the cells were harvested and resuspended with 300 µl of extraction buffer (25 mM glycylglycine, 15 mM MgSO, 4 mM EGTA, 1% Triton X-100, and 1 mM dithiothreitol) and assayed for luciferase activity(26) . Transfection efficiency was monitored by a thymidine kinase (TK) human growth hormone (GH) expression plasmid (TKGH). GH levels were measured in 50 µl of media by chemiluminescent assay (Nichols Institute, San Juan Capistrano, CA). Luciferase expression was quantified as arbitrary light units/µl of extraction buffer.

Gel Mobility Shift Assays

Double-stranded DNA probes representing PAL (5`-TCAGGTCATGACCTGAC-3`) and DR+4 (5`-TCAGGTCACTTCAGGTCAAC-3`) were radiolabeled using the polymerase chain reaction and [P]dCTP (400 µCi/m). Unincorporated P was removed by G-25 Sephadex chromatography. The TR cDNAs were cloned into PGEM 3Z and the RXR cDNA was cloned into pBluescript (a gift of R. Evans, Salk Institute). Proteins were made by in vitro translation in rabbit reticulocyte lysate (TNT, Promega) using either T7 or T3 polymerase. To quantify protein production, S incorporation and direct visualization on SDS-polyacrylamide gel electrophoresis (12%) was utilized. 3 µl of each protein was used in each reaction with radiolabeled probe (75-100,000 cpm). Binding reactions were performed in a 20-µl volume of binding buffer (20% glycerol, 20 mM HEPES, pH 7.6, 50 mM KCl), 1 mM dithiothreitol, 1 µg of poly(dI-dC), 0.1 µg of salmon sperm DNA. Incubations were carried out at room temperature for 30 min. Gel mobility shifts were resolved on 5% non-denaturing polyacrylamide gels and visualized after autoradiography.

Whole cell extracts were made from COS-1 cells transfected as described above with 10 µg of expression plasmid per 100-mm plate. After 48 h, the cell cultures were harvested and resuspended in 25 µl of binding buffer containing 1 mM of phenylmethylsulfonyl fluoride and 1 mM of dithiothreitol. Extracts were prepared by three freeze-thaw cycles.


RESULTS

Human TR1 Is More Active in Both Ligand-independent and Ligand-dependent Function on Positive TREs

To evaluate the relative activity of the TR isoforms on positive TREs, CV-1 cells were cotransfected with equal amounts of TR isoform expression vectors and two separate positive TREs linked to a luciferase reporter, DR+4 and PAL (see ``Materials and Methods''). In the absence of ligand, TR1 was over 2-fold more potent than TR1 as a transcriptional silencer on the DR+4 element (6.6- versus 2.6-fold) (Fig. 1A) and approximately 2-fold more potent in ligand-independent activity on the PAL element (7.2- versus 3.7-fold). Similar isoform potency effects were seen in the presence of 10 nM T, where TR1 was over 2-fold more potent in its ability to activate transcription on both PAL and DR+4 (45- versus 20-fold and 9- versus 4.4-fold, respectively) (Fig. 1B).


Figure 1: Ligand-independent and -dependent effects of TR isoforms on positive TREs is isoform specific. A, CV-1 cells were cotransfected with PAL and DR+4 TREs either in the presence of TR isoform or PKCR-2 alone. The data are presented as fold repression from basal expression (PKCR-2 alone) and are the mean of three separate experiments. The errorbars indicate standard error of the mean (S.E.). B, CV-1 cells were cotransfected with PAL and DR+4 and either TR isoform in the presence or absence of 10 nM T. The data is presented as fold stimulation (10 nM T) over basal (in the presence of TR isoform alone).



To control for relative levels of TR isoform expression, we 1) transfected increasing amounts of TR isoform in the presence of both TREs and T and 2) isolated cellular extracts from transfected COS-1 cells (which are deficient in endogenous RXR(27) ) and assessed relative amounts of protein produced in gel mobility shift assays. Fig. 2A demonstrates that the potency differences between the isoforms, in the presence of T, is not due to increasing amounts of transfected TR1 versus TR1. Ligand-independent repression was preserved at all doses of receptor used. The receptor dose chosen for all future experiments (80 ng/well) gave near maximal ligand-independent and -dependent effects on both reporters studied.


Figure 2: Isoform effects are independent of transfection efficiency. A, CV-1 cells were cotransfected with either PAL or DR+4 (1.6 µg/well) and increasing amounts of TR isoform in the presence or absence of 10 nM T. The data are presented as fold stimulation ± S.E. in the presence of 10 nM T. The solidbars represent TR1, and the closedbars represent TR1. B, 5 µl of COS-1 cell whole cell extracts of TR isoforms, chimeras and mutants, and PKCR-2 were incubated with 3 µl of in vitro translated RXR and radiolabeled DR+4. The arrows identify the specific bands. The indicated probe is shown below the autoradiograph. NS, nonspecific band.



Quantification of TR isoform expression was performed in cellular extracts (Fig. 2B) from the CV-1-related cell line COS-1 and supports the above data. A relative increase in TR1 isoform-RXR heterodimer is formed with reticulocyte lysate-produced RXR and whole cell extract-produced TR. As TR1 function is relatively preserved over a wide range of receptor concentrations (Fig. 2A), receptor dose is not responsible for the potency differences observed. The mobility of the heterodimers produced are consistent with the different sizes of the human TR isoforms (55 kDa for TR1 and 46 kDa for TR1) (28, 29) due primarily to their different sized amino termini.

The Amino Terminus of TR1 Is Responsible for the Increased Function of TR1 on Positive TREs

A number of chimeric and mutated receptors were constructed to define the functional domain of TR1, which was responsible for its increased ligand-independent and -dependent function on DR+4 and PAL (Fig. 3). These chimeric receptors were used in an identical cotransfection paradigm as described above. As Fig. 4A demonstrates, the addition of the TR1 amino terminus to the DNA and ligand binding domains of TR1 (TR, ) causes a reconstitution of the wild-type TR1 effect on PAL, both in the presence and absence of ligand. Conversely, swapping the TR1 amino terminus with the amino terminus of TR1 to form TR causes ligand-independent and -dependent function to decrease to levels found with TR1 on PAL. Deletion of the TR1 and TR1 amino termini (TR and TR, respectively) leads to receptors that possess significantly less ligand-independent and -dependent activity than their wild-type originators on PAL.


Figure 3: Structure of TR isoform chimeras and mutants. A number of TR chimeras and mutants were created using restriction enzyme digestion and polymerase chain reaction. The numbering indicates the amino acid number in each isoform. The designation of each chimera with threesymbols indicates the origin of the amino terminus, DNA binding domain, and ligand binding domain.




Figure 4: Isoform specificity resides in the amino terminus. A, CV-1 cells were cotransfected with PAL and the various TR constructs in both ligand-independent and -dependent paradigms as described in Fig. 1. The data are reported as % maximal fold repression (where %100 equals the activity of TR1 (see Fig. 1)) for ligand-independent effect and % maximal fold stimulation for ligand-dependent (10 nM T) effect. The data are the mean of three separate experiments and are displayed with the S.E. B, CV-1 cells were transfected as above with DR+4 as the reporter. The data are displayed as above and are the mean of three separate experiments.



Similar data are achieved in our analysis on DR+4 (Fig. 4B). TR is approximately 2-fold more active than TR1 in ligand-independent and -dependent function on DR+4 and has 75% of the effect of TR1 in both of these functions. In contrast, TR is more similar in both ligand-independent and -dependent activity to TR1 (it is in fact less active). However, TR is more potent in both ligand-independent and ligand-dependent properties than TR, suggesting that the DNA binding domain and the hinge region may play some role in isoform specificity on this element consistent with their well defined role in half-site recognition and heterodimerization(30) . This is supported by the fact that although deletion of the amino termini of both TR isoforms leads to a significant loss of function, TR is still more active than TR in all respects on DR+4.

The Amino Terminus of TR1 Inhibits Homodimer Formation on DR+4 and PAL

To examine whether the amino terminus of TR1 could influence DNA binding, we performed gel mobility shift assays with the wild-type, mutated, and chimeric receptors. Analysis of in vitro translated receptor produced was performed by [S]methionine incorporation and direct visualization (Fig. 5). The size differences and presence of two translation products of the various receptors correspond to previous work(28, 29) , and their known sizes (55 kDa for TR1 and 46 kDa for TR1). The size of the chimeras are dictated by the origin of their amino terminus. TR is smaller than all of the chimeric constructs and shows only one translation product because of the deletion of its entire amino terminus and the insertion of a single translational start site. In the in vitro translation system used, TR1 amino terminus containing receptors are produced at approximately 25% of the level of TR1 amino terminus containing receptors (Fig. 5, first and lastlanes). When the TR1 translation start site was converted to a Kozak consensus site, translation efficiency was not improved (data not shown). In contrast, this same Kozak site was used in generating TR and TR where translation of both proteins is enhanced relative to their respective full-length receptors. This suggests that the quantity of TR1 produced in vitro is intrinsic to the amino terminus of the receptor itself. Therefore, initial examination of the ability of TR1 to homodimerize was performed using increasing amounts of the in vitro translated product.


Figure 5: In vitro translation of TR isoforms, chimeras, and mutants. The different TR constructs were in vitro translated in reticulocyte lysate, and the translation products were quantified by direct incorporation of [S]methionine and then visualized on SDS-polyacrylamide gel electrophoresis. The arrows demonstrate the relative location of the TR proteins (dashedarrow, TR1; solidarrow, TR1. Receptors containing either the TR1 or TR1 amino termini have two translation products, while the truncated TR contains only one (see text).



As shown in Fig. 6, increasing amounts of TR1 leads to the formation of a single migrating complex with increased mobility, which represents monomer on both PAL and DR+4 (see Fig. 7for comparison of distances of migrating complexes). The addition of RXR leads to the formation of a strong heterodimer. Despite using up to four times the amount of lysate used in other gel mobility shifts, no homodimerization of TR1 was seen.


Figure 6: TR1 preferentially heterodimerizes on PAL and DR+4. Gel mobility shift assay of increasing amounts of in vitro translated TR1 as indicated and a constant amount of RXR with both radiolabeled DR+4 and PAL as probes. HD, heterodimer; M, monomer; NS, nonspecific band.




Figure 7: The amino terminus of TR1 antagonizes homodimerization on DR+4 and PAL. A, gel mobility shift assay of radiolabeled DR+4 incubated with in vitro translated TR constructs and RXR. The concentration of T used is 10 nM. Lane1 contains unprogrammed reticulocyte lysate. B, gel mobility shift assay of radiolabeled PAL with the same in vitro translated proteins as above. C, gel mobility shift assay of radiolabeled PAL with in vitro translated and RXR with wild-type TR used as a control. HD, heterodimer; D, homodimer; M, monomer; NS, nonspecific band.



When DR+4 was used as a probe with the chimeric and mutated receptors, the TR1 amino terminus inhibited homodimer formation (Fig. 7A) as TR only formed a heterodimeric complex with RXR while TR leads to the formation of homo- and heterodimeric complexes. In contrast, the TR amino terminus facilitates homodimerization as TR forms a strong homodimer, unlike TR1 (Fig. 6). Interestingly, the deletion of the amino terminus of TR1 (TR) causes a loss of heterodimeric capability on DR+4 and favors the formation of a strong homodimer. A similar loss in heterodimeric capability was seen when the amino terminus of TR1 (TR) was deleted (data not shown). These data indicate that while both amino termini antagonize homodimer formation, the TR1 terminus is much more potent in this regard.

Similar DNA binding specificity was observed on the PAL probe (Fig. 7B). However, the PAL element is more amenable to heterodimerization than DR+4 (31) such that the preference of the amino terminus of TR1 to allow for heterodimerization is increased. Still, exchanging the amino termini of TR1 and TR1 (see TR versus TR) dramatically changes the relative ratios of hetero- versus homodimer that is not related to amount of receptor protein. TR forms both a weaker homodimer on PAL than DR+4 and, in contrast to its activity on DR+4, a weaker homodimer than TR1 on PAL. TR was able to form a strong homodimer and heterodimer on PAL (Fig. 7C), consistent with the TR1 amino terminus antagonizing homodimer formation.


DISCUSSION

We have used transient transfection in mammalian cells and gel mobility shift assays to further explore the role of TR isoform specificity on gene regulation. From our data, it is clear that TR1 preferentially acts as a ligand-independent silencer and ligand-dependent activator on the positive TREs studied. These studies also further establish the important role of ligand-independent repression on the relative level of target gene expression either in the presence or absence of T. It is also clear that the distinct amino terminus of TR1 allows for the isoform-specific effects on both DR+4 and PAL. Inclusion of the TR1 amino terminus in TR chimeras allows for 2-fold increases in both ligand-independent and -dependent functions on DR+4 and PAL. The TR1 amino terminus also inhibits homodimerization on DR+4 and PAL, indicative of the fact that the relative changes in binding specificity may underlie the functional effects seen (Fig. 8).


Figure 8: Model relating DNA binding to functional activity. The wild-type, chimeric, and mutant receptors are shown in cartoon form as either homodimers (opencircle) or as heterodimers with RXR (opensquare). The arrow indicates the favored conformation of the particular receptor shown. The relative functional activity of each of the receptors in the presence or absence of ligand is listed above.



The mechanism of ligand-independent silencing is not clear but is presumed to result from the interaction of the TR with other proteins, such as TFIIB(32, 33) . Recent data suggest that the TRRXR heterodimeric complex is responsible for this ligand-independent effect, as TR mutants that are defective in their ability to homodimerize but still retain the ability to heterodimerize either retain or are enhanced in their ability to act as transcriptional silencers(13) . Also, the TR homodimer, which only forms in the presence of a DNA binding site(34) , may compete with the heterodimer, in the absence of T, for TRE binding. In this case, enhanced homodimerization would lead to less silencing as these complexes may be transcriptionally inactive(35) . One region of the TR specifically involved in ligand-independent activity may lie in the hinge region (36) of the TR in both isoforms as mutagenesis of a proline (amino acid 209 in TR1 and amino acid 160 in TR1) causes a specific loss of ligand-independent activity but preserves ligand-dependent function. Thus, in addition to other previously characterized regions of the TR important for heterodimerization(37, 38) , the TR1 amino terminus by enhancing heterodimeric capability or inhibiting homodimerization allows for increased ligand-independent function on certain TREs. Whether this is due entirely to DNA binding or to the exposure of a new activation domain in the amino terminus remains to be determined.

A number of lines of evidence also support the hypothesis that TRRXR heterodimerization is essential for ligand-dependent transcriptional activation. 1) T binding dissociates TR homodimers from positive TREs (39) (see Fig. 7A), allowing preferential binding of TRRXR heterodimers; 2) TRRXR heterodimers have a stronger affinity for TREs than TR homodimers(8) ; 3) cotransfection of RXR enhances the positive transcriptional response to T on a number of different elements(10, 40) ; and 4) more recently yeast expression systems demonstrate that TRRXR heterodimerization is necessary for a full ligand-dependent transcriptional response(41) . The data presented here support this hypothesis by demonstrating that the propensity for a receptor to heterodimerize is influenced strongly by its amino terminus and determines its ability to respond to ligand. Similar to their ligand-independent effects, TR mutants that cannot homodimerize are still powerful ligand-dependent transcriptional activators (13), which supports the notion that propensity to heterodimerize determines ligand-dependent transcriptional ability. Further work in this area will likely focus on the mechanism by which the TRRXR complex is able to cause ligand-independent and -dependent effects. Specific co-activators are likely to be involved, such as TFIIB (32, 33) and other unidentified proteins, which may interact with the TRRXR complex.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK-43653 (to F. E. W.) and K08D4-02354 (to A. N. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Thyroid Unit, Beth Israel Hospital, Research North 330B, 330 Brookline Ave., Boston, MA 02215. Fax: 617-735-2927.

The abbreviations used are: TREs, thyroid hormone response elements; RXR, retinoid X receptor; TR, thyroid hormone receptor; T, triiodothyronine.


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