Transcriptional Repression by Thyroid Hormone Receptors

A ROLE FOR RECEPTOR HOMODIMERS IN THE RECRUITMENT OF SMRT COREPRESSOR*

Sunnie M. Yoh and Martin L. PrivalskyDagger

From the Section of Microbiology, Division of Biological Sciences, University of California, Davis, California 95616

Received for publication, November 3, 2000, and in revised form, January 17, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear hormone receptors, such as the thyroid hormone receptors (T3Rs) and retinoid X receptors (RXRs), are ligand-regulated transcription factors that control key aspects of metazoan gene expression. T3Rs can bind to DNA either as receptor homodimers or as heterodimers with RXRs. Once bound to DNA, nuclear hormone receptors regulate target gene expression by recruiting auxiliary proteins, denoted corepressors and coactivators. We report here that T3R homodimers assembled on DNA exhibit particularly strong interactions with the SMRT corepressor, whereas T3R·RXR heterodimers are inefficient at binding to SMRT. Mutants of T3R that exhibit enhanced repression properties, such as the v-Erb A oncoprotein or the T3Rbeta -Delta 432 mutant found in human resistance to thyroid hormone syndrome, display enhanced homodimerization properties and exhibit unusually strong interactions with the SMRT corepressor. Significantly, the topology of a DNA binding site can determine whether that site recruits primarily homodimers or heterodimers and therefore whether corepressor is efficiently or inefficiently recruited to the resulting receptor-DNA complex. We suggest that T3R homodimers, and not heterodimers, may be important mediators of transcriptional repression and that the nature of the DNA binding site, by selecting for receptor homodimers or heterodimers, can influence the ability of the receptor to recruit corepressor.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Small, hydrophobic hormones, such as steroids, retinoids, and the thyroid hormones T3 and T4 thyronine, regulate many important aspects of metazoan differentiation, reproduction, and homeostasis. These hydrophobic hormones function by binding to specific nuclear receptors that operate as hormone-regulated transcription factors (1-6). Different nuclear receptors bind to different DNA sequences, denoted hormone response elements (HREs),1 and modulate the expression of adjacent target genes. In this fashion, different sets of target genes are regulated in response to different hormone ligands, leading to physiologically appropriate changes in target cell phenotype (1-3, 5, 6).

Many nuclear hormone receptors possess bipolar transcriptional properties and can either repress or activate expression of target promoters (7-9). These bimodal regulatory properties are manifested through the ability of nuclear receptors to interact with auxiliary proteins, denoted corepressors and coactivators (reviewed in Refs. 10-14). Thyroid hormone receptors (T3Rs) and retinoic acid receptors (RARs), for example, typically interact with a corepressor complex in the absence of hormone and thereby confer transcriptional repression (15-22). The corepressor complex includes SMRT or its paralog N-CoR, which contacts the nuclear receptors directly, as well as additional proteins that are tethered indirectly through interactions with SMRT, such as mSin3, SAPs, Ski, and an assortment of histone deacetylases (23-32). The addition of cognate hormone induces a conformational change in the T3Rs and RARs, leading to dissociation of the SMRT corepressor complex from the receptor and recruitment of a novel set of coactivator proteins that confer transcriptional activation (33-36). Coactivators include the p160 family, such as SRC-1 and GRIP-1, CBP/p300, and the DRIP-TRAP-SMCC complex (10-14, 37, 38). Once recruited, coactivators and corepressors regulate transcription through modification of the chromatin template and by interactions with the general transcriptional machinery (12-14, 23, 25, 39-42).

Nuclear receptors bind to DNA with each receptor molecule recognizing a conserved, 6-8-base DNA sequence referred to as a "half-site" (3). Given that most nuclear receptors are able to bind to DNA as protein dimers, a prototypic HRE is composed of two half-sites (43, 44). The sequence of the individual half-sites, their spacing, and their orientation contribute to the specificity of DNA recognition by the different nuclear receptors (45-47). Notably, however, a surprisingly diverse array of DNA sequences can serve as response elements for a given nuclear receptor. T3Rs, for example, are able to bind to HREs comprised of two half-sites oriented as a direct repeat with a 4-base spacer (DR-4), an inverted repeat with no spacer (INV-0), or a divergent repeat with a 6-base spacer (DIV-6) (46-52). Imposed on this diversity of HREs is the ability of many nuclear receptors to bind to DNA not only as homodimers but also as heterodimers with other members of the nuclear receptor family. Retinoid X receptors (RXRs) are particularly important heterodimer partners for many nuclear receptors, and T3R·RXR and RAR·RXR heterodimers can form with higher affinity and exhibit stronger transcriptional activation properties than do the corresponding homodimers (3, 4, 45).

We wished to determine if the ability of nuclear receptors to recruit SMRT corepressor might differ for receptor homodimers versus heterodimers and if the nature of the DNA response element could influence, directly or indirectly, this receptor/corepressor interaction. We report here that T3R homodimers assembled on DNA exhibit particularly strong interactions with the SMRT corepressor, whereas T3R·RXR heterodimers are inefficient at binding to corepressors. Mutants of T3R that exhibit enhanced repression properties, such as the v-Erb A oncoprotein or the T3Rbeta -Delta 432 mutant found in human resistance to thyroid hormone (RTH) syndrome (8, 53), display enhanced homodimerization properties and exhibit unusually strong interactions with corepressor. Significantly, the orientation and spacing of the half-sites in a response element can influence whether a given response element recruits v-Erb A primarily as a homodimer or as a v-Erb A/RXR heterodimer and therefore whether corepressor is efficiently or inefficiently recruited to the v-Erb A-DNA complex. Transfection experiments further support the suggestion that transcriptional repression may be mediated primarily through the actions of receptor homodimers rather than heterodimers with RXR. In contrast to T3Rs, however, RARs efficiently recruit corepressor both as homodimers and as heterodimers with RXR, indicating that the nature of the receptor dimer may play a less decisive role in corepressor recruitment by RARs.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Clones, Oligonucleotides, and Antisera----- The pSG5 mammalian expression vectors have been described previously and include the wild-type and Delta 432 mutant T3Rbeta clones (53), the wild-type avian T3Ralpha and v-ErbA clones (54), and the human RARalpha and RXRalpha clones (34). The baculovirus expression vectors for T3Ralpha , T3Rbeta , RARalpha , and RXRalpha were also detailed in a previous publication (55). For expression as a glutathione S-transferase (GST) fusion protein in bacteria, a suitable restriction fragment representing amino acids 1056-1495 of SMRT was inserted into the pGEX-KG vector and transfected into Escherichia coli strain DH5alpha (53, 56). RXR-directed antiserum was a generous gift from Pierre Chambon (INSERM, Strasborg, France). The oligonucleotides used in the DNA binding assays were as follows: DR-4, 5'-TCGAATAAGGTCAAATAAGGTCAGAG-3'; DIV-6, 5'-TCGATACGATCGTGACCTATTAGGAGGTCAACAGACGGG-3'; INV-0, 5'-TCGAGTTCTCAGGTCATGACCTGAGAAC-3'; rGH, 5'-TCGAGGAAAGGTAAGATCAGGGACGTGACCGCAGGAG-3'; cLYS, 5'-TCGAATTATTGACCCCAGCTGAGGTCAAGTTACG-3'; DR-5, 5'-TCGACTCTGACCTCTCGTTGACCTGCT-3'; biotin-DR-4, 5'-XTCGAATAAGGTCAAATAAGGTCAGAGTCTGA-3'; biotin-DR-4mut, 5'-XTCGAATAAGATCAAATAAGATCAGAGTCGA-3'.

Electrophoretic Mobility Shift Assays-- T3Ralpha , T3Rbeta , RARalpha , and RXRalpha were prepared by expression in a recombinant baculovirus/Sf-9 cell system (55). GST and GST-SMRT proteins were expressed in E. coli and were purified by glutathione-agarose affinity chromatography (53); the resulting proteins were reconstituted at a concentration of 2-10 mg/ml in 50 mM Tris, pH 7.4, 20% glycerol, 200 µg/ml bovine serum albumin. Electrophoretic mobility shift assays (EMSAs) were performed by mixing 1-2 ng of each nuclear receptor preparation with a 32P-radiolabeled oligonucleotide probe (40,000-60,000 cpm, representing 20-60 ng of DNA) at 25 °C for 25 min in 15 µl of binding buffer (10 mM Tris-Cl, pH 7.5, 3% glycerol, 66.7 mM KCl, 2 mM MgCl2, 13.3 µg/µl bovine serum albumin, 0.113 µg/µl poly(dI-dC). The resulting DNA-protein complexes were resolved by nondenaturing gel electrophoresis through a 5% polyacrylamide gel (30:1 acrylamide/bisacrylamide); the free and bound DNA probe was visualized by PhosphorImager analysis (STORM system; Molecular Dynamics, Inc., Sunnyvale, CA) and was quantified using ImageQuant software (Molecular Dynamics). For supershift experiments using corepressor, the nuclear receptor preparations were preincubated with either GST or GST-SMRT for 10 min on ice prior to the addition of the oligonucleotide probe. For antibody supershifts, the protein/DNA complexes were allowed to form for 15 min at 25 °C, and then suitable antisera were added, and the incubation continued for an additional 15 min at 25 °C prior to gel electrophoresis.

EMSAs were also performed using nuclear receptors synthesized from pSG5 vectors by use of a coupled in vitro transcription/translation system (TnT system; Promega). A total of 6 µl of each in vitro synthesized receptor preparation was incubated with the radiolabeled oligonucleotide probe (100,000 cpm, representing 100 ng of DNA) for 20 min at 25 °C in 40 mM HEPES, pH 7.8, 50 mM KCl, 5 mM MgCl2, 1 µM ZnCl2, 6% glycerol, 0.2 µg/µl bovine serum albumin, 0.066 µg/µl poly(dI-dC). The resulting protein/DNA complexes were then resolved by electrophoresis on a 5% polyacrylamide gel containing 1% glycerol and were visualized and quantified as above.

Avidin-Biotin-DNA Binding Assay-- Approximately 0.05 µg of each biotin-tagged oligonucleotide were immobilized by incubation with 20 µl (packed volume) of streptavidin-agarose. The oligonucleotide-agarose complexes were then incubated for 30 min at 4 °C with 250 ng of T3Rbeta , or of T3Rbeta and RXRalpha , in 500 µl of PBST buffer (150 mM NaCl, 16 mM Na2HPO4, 4 NaH2PO4, 2 mM EDTA, 1 mM dithiothreitol, and 1% Triton X-100) supplemented with 10 mg/ml bovine serum albumin and 1× Complete protease inhibitor (Roche Molecular Biochemicals). The agarose-DNA-receptor complexes were then washed three times with 1 ml of PBST buffer and once with 1 ml of HEMG buffer (53) prior to incubation with 5 µl of 35S-labeled, in vitro transcribed/translated SMRT protein. The agarose DNA-protein complexes were finally washed four times with 1 ml of HEMG buffer, and the proteins were eluted in SDS sample buffer. The eluted proteins were resolved by SDS-polyacrylamide gel electrophoresis and were characterized by Western and PhosphorImager analyses (57).

Transient Transfections-- CV1 cells were transfected by use of a Lipofectin protocol (Life Technologies, Inc.). Typically 50 ng of the pSG5-T3Ralpha vector was used per well of a 12-well plate (~5 × 104 cells), together with 200 ng of a thymidine kinase promoter-luciferase reporter containing a DR-4 element, 200 ng of pCH110-lacZ (employed as an internal control), and various amounts of a pSG5-RXRalpha expression vector (53).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

SMRT Corepressor Preferentially Interacts with Homodimers, Not Heterodimers, of T3R on DR-4 DNA Response Elements-- We and others have reported that SMRT interacts more avidly with nuclear receptor dimers than with receptor monomers (16, 58-60). However, most of these prior studies employed two-hybrid and GST pull-down assays and did not address the corepressor interaction properties of defined receptor complexes assembled on authentic DNA response elements. We therefore employed an EMSA to determine the ability of T3R alone or of RXR and T3R together to interact with SMRT when these receptors were bound to a suitable DNA response element. We first examined the ability of T3R or T3R·RXR to bind to a DR-4 DNA element and the ability of SMRT to "supershift" the mobility of the resulting receptor-DNA complexes. T3Ralpha formed a single predominant complex when incubated with the radiolabeled DR-4 probe (Fig. 1A, denoted T3R·T3R). This protein-DNA complex displayed all of the characteristics previously noted for a T3R homodimer: the protein-DNA complex migrated at an appropriate electrophoretic mobility for a homodimer, it was supershifted with anti-T3R antibodies but not with RXR-directed antibodies, the presumptive homodimer complex was destabilized by the addition of T3 hormone (61), and no protein-DNA complex was observed using otherwise identical control lysates of nonrecombinant baculovirus/SF9 cells (Fig. 1A). The addition of RXR to the T3R preparation resulted in the formation of a novel complex exhibiting the properties of a T3R·RXR heterodimer (Fig. 1A, denoted T3R/RXR). This complex migrated at a slower mobility than that of the T3R·T3R complex, was stable to T3 hormone (61), and could be supershifted with either anti-T3R or anti-RXR antibodies. RXR alone failed to bind at significant levels to the DR-4 DNA probe under these conditions (Fig. 1A).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 1.   Preferential interaction of SMRT corepressor with T3Ralpha homodimers on a DR-4 DNA response element. A, T3Ralpha binds to a DR-4 DNA element either as T3R·T3R homodimers or as T3R·RXR heterodimers. Either 2 ng of T3Ralpha alone, 2 ng of RXRalpha alone, or 1 ng each of T3Ralpha and human RXRalpha together were incubated with a 32P-labeled DR-4 DNA probe at 25 °C for 15 min in either the presence or absence of T3, as indicated below. Antiserum directed against RXR (X), T3R (T), or normal rabbit serum (N) was also introduced to several of the receptor/DNA mixtures as indicated below. The resulting protein-DNA complexes were resolved by gel electrophoresis and were visualized by PhosphorImager analysis. A nonrecombinant baculovirus preparation was employed as a negative control (lane 1). Complexes corresponding to the T3R·T3R homodimer and to the T3R·RXR heterodimer are indicated to the right; the corresponding protein-DNA complexes, supershifted by antisera, are indicated with asterisks. The portion of the electrophoretogram representing free probe has been omitted to permit a clearer display of the protein-DNA complexes; the DNA probe was in excess in all cases. B, SMRT interacts efficiently with T3Ralpha ·T3Ralpha homodimers but not with T3Ralpha ·RXRalpha heterodimers, bound to a DR-4 DNA element. A similar analysis was performed as in A, except GST (-) or GST-SMRT (+) proteins were also included in the DNA binding reaction, as indicated below. The positions of the T3R·T3R, T3R·RXR, and T3R·T3R·SMRT complexes are indicated to the right. Nonrecombinant baculovirus/Sf-9 lysates were employed as a negative control in lanes 9 and 10 (No). C, the T3R·T3R·SMRT complex contains T3R but not RXR. The receptor-SMRT-DNA complex analyzed in B was incubated with either normal serum, T3R-specific antiserum, or RXR-specific antiserum, denoted as in A. Protein-DNA complexes supershifted by antisera are indicated with asterisks.

A GST-SMRT derivative, purified from E. coli, did not detectably bind to the DR-4 probe when tested alone (Fig. 1B, lane 10). However, the addition of the GST-SMRT derivative to the T3R protein preparation resulted in a further reduction in the electrophoretic mobility of the T3R homodimer complex (i.e. a "supershift") indicative of an SMRT/T3R interaction (Fig. 1B, lane 3; labeled T3R/T3R/SMRT). No supershift was observed with a nonrecombinant GST preparation (Fig. 1B, lane 1). As anticipated for an authentic SMRT·T3R complex, antibodies to T3R, but not to RXR, further shifted the migration of the T3R·T3R·SMRT complex to a still slower mobility, and the supershifted T3R·T3R·SMRT species could be dissociated by the addition of T3 hormone (Figs. 1, B and C) (20).

In contrast to the robust supershift observed for T3R·T3R homodimers, the addition of GST-SMRT to T3R·RXR heterodimers assembled on the same DR-4 DNA element resulted in a very weak supershift, with the bulk of the heterodimer failing to detectably interact with the corepressor (Fig. 1B, lanes 5-8; quantified in Fig. 2A). The relatively poor supershift of the T3R·RXR heterodimer compared with the T3R·T3R homodimer was observed under a variety of conditions, using a range of GST-SMRT concentrations, and was not enhanced by the addition of an RXR-specific ligand; the DNA probe was in excess in all experiments (data not shown). Of note, the modest amount of SMRT-supershifted complex that did form in the presence of T3R·RXR heterodimers reacted primarily with anti-T3R but not with anti-RXR antibodies (see below); these results suggest that even in the presence of a T3R·RXR heterodimer, the corepressor may be able to recruit T3R homodimers, perhaps by shifting the receptor equilibrium away from the heterodimer toward the homodimeric species.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 2.   Selectivity of SMRT for homodimers, not heterodimers, of T3R. A, quantification. The results in Fig. 1B were quantified by PhosphorImager analysis. The amount of receptor-DNA-SMRT complex formed (black bars) is displayed relative to the total amount of receptor-DNA complex (stippled bars). The amount of receptor-DNA complex formed in the absence of SMRT and T3 was defined as 100%. B, a similar analysis was performed as in Fig. 1, B and C, except the amount of RXRalpha was titrated so as to yield a mixed population of T3Ralpha ·T3Ralpha homodimers and T3Ralpha ·RXRalpha heterodimers (lane 7). This mixed receptor population was challenged by the addition of GST-SMRT (lanes 8 and 10), GST alone (lanes 7 and 9), and/or an antibody against RXR (lanes 9 and 10). Lanes 1-6 represent a comparison experiment performed in an identical fashion on T3Ralpha ·T3Ralpha homodimers in the absence of RXR.

Our results suggested that T3R homodimers, when bound to a DR-4 element, interacted more strongly with corepressor than did T3R·RXR heterodimers. To more rigorously test this hypothesis, we employed the DR-4 DNA element again and adjusted our EMSA conditions to generate a mixed receptor population, so that both T3R·T3R homodimers and T3R·RXR heterodimers were present in the same reaction (Fig. 2B, lane 7). We then challenged this mixed receptor population by the addition of the GST-SMRT construct. Under these conditions, virtually all of the T3R·T3R homodimer was supershifted into the T3R·T3R·SMRT complex, whereas very little of the T3R·RXR heterodimer, present in the same reaction, was supershifted by the addition of GST-SMRT (Fig. 2B, lane 8). Close inspection did reveal that introduction of SMRT under these conditions resulted in a modest reduction in the amount of T3R·RXR heterodimer (compare lane 8 with lane 7); nonetheless, the predominant SMRT-containing supershifted complex even under these conditions was the T3R·T3R·SMRT species (compare lane 8 with lane 3), and this supershifted complex did not contain significant amounts of RXR when probed with RXR-directed antibodies (compare lane 10 with lanes 8 and 9). These results support the hypothesis that SMRT preferentially binds to T3R·T3R homodimers, not to T3R·RXR heterodimers, and indicate that this preferred binding of the homodimer by SMRT may be sufficient to partially shift the receptor equilibrium away from the heterodimer species.

The Preferential Interaction of SMRT Corepressor with T3Ralpha Extends to Other Receptor Isoforms and Was Also Detected by an Avidin-Biotin Protocol-- In addition to the T3Ralpha tested above, a second, T3Rbeta , locus is present in all vertebrates (1, 2). T3Rbeta exhibits a distinct expression pattern from that of T3Ralpha and plays a distinct, if partially overlapping, role in organismal development and physiology. In common with our results with T3Ralpha , T3Rbeta homodimers bound to a DR-4 DNA element and interacted readily with SMRT to generate a T3R·T3R·SMRT complex, whereas T3Rbeta ·RXR heterodimers, assembled on the same DR-4 element, interacted with SMRT with much lower efficiency (data not shown). Similarly, RXRs are encoded by three different loci: RXRalpha , -beta , and -gamma (4). Use of the RXRbeta or RXRgamma isoforms instead of RXRalpha did not alter the relative inability of the T3R·RXR heterodimer to interact with SMRT (data not shown). Therefore, the preferential interaction of SMRT with T3R homodimers, rather than with T3R·RXR heterodimers, extends to both of the major T3R isoforms and to all three of the major RXR isoforms.

Our results, obtained by EMSA on receptors bound to DNA, appeared to contradict studies, using GST pull-down or two-hybrid approaches, that suggested T3R interacts with SMRT more strongly in the presence of RXR than in its absence (58, 60). We therefore confirmed our results by use of an alternative methodology. We incubated T3Rbeta -1 with a biotinylated, but otherwise unlabeled, DR-4 DNA element. The proteins binding to the DNA probe were subsequently isolated by adsorption of the biotinylated DNA to a streptavidin matrix. The DNA-protein-streptavidin complexes were washed, and the proteins were eluted and were resolved by SDS-polyacrylamide gel electrophoresis. The proteins that bound to the DNA element were then visualized by using either a radiolabeling or immunoblotting procedure. As expected, little or no T3R bound to the streptavidin matrix in the absence of DNA or if a biotinylated DR-4 probe bearing a dysfunctional, mutated response element sequence was employed (Fig. 3A, lanes 1, 4, and 5). In contrast, significant amounts of T3R bound to a biotinylated DNA probe representing a DR-4 HRE (Fig. 3A, lanes 2 and 3; the T3R homodimer was not detectably destabilized by T3 hormone when assayed in this fashion, probably reflecting minor technical differences between the avidin-biotin and the EMSA approaches). The addition of RXR increased the amount of T3R bound by the biotinylated probe, and the RXR protein itself was now also found in the biotinylated DNA complex, consistent with formation of a T3R·RXR heterodimer (Fig. 3B; quantified in Fig. 3C). Consistent with our EMSA experiments, the T3R complexes formed on the biotinylated DR-4 element in the absence of RXR were able to interact with the corepressor, resulting in copurification of SMRT on streptavidin-agarose (Fig. 3, B and C). Conversely, although the addition of RXR greatly increased the amount of nuclear receptors bound to the biotinylated DR-4 probe, the resulting T3R·RXR complexes displayed a significantly reduced ability to bind to SMRT compared with the T3R homodimer complexes (Figs. 3, B and C). Of note, several SMRT variants have been described (e.g. Refs. 15, 19, 20). The 1495-codon SMRT version was used for Fig. 3; comparable results, however, were obtained with a longer, 2517-codon form of SMRT (data not shown). We conclude that, both by EMSA and by avidin/biotin protocols, the T3R homodimer recruits SMRT more efficiently than does the T3R·RXR heterodimer.


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 3.   Preferential interaction of SMRT corepressor with T3Rbeta homodimers detected by an avidin-biotin assay. A, T3Rbeta complexes assemble in a sequence-specific fashion on biotinylated DNA response elements. Biotin-conjugation DR-4 oligonucleotides representing either the wild-type DR-4 sequence (WT) or a mutated DR-4 sequence (mut) were immobilized on streptavidin-agarose and were incubated with 35S-labeled T3Rbeta in the absence or presence of T3 hormone, as indicated below. The receptor-DNA-agarose complexes were then washed, the proteins remaining bound to the DNA-agarose matrix were eluted, and the eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis. As an additional negative control, the ability of the radiolabeled T3Rbeta to bind to streptavidin-agarose in the absence of any oligonucleotides was also tested (lane 1). B and C, homodimers of T3Rbeta recruit corepressor SMRT more efficiently than do heterodimers of T3Rbeta /RXRalpha . Either T3Rbeta alone or T3Rbeta and RXRalpha together, as indicated, were incubated with the immobilized wild-type DR-4 oligonucleotide as in A. The resulting receptor-DNA-agarose complexes were further incubated with 35S-labeled SMRT. The protein-DNA complexes were then washed, eluted from the DNA-agarose matrix, and resolved by SDS-polyacrylamide gel electrophoresis. The electrophoretograms were visualized by immunoblotting and/or PhosphorImager analysis (B), and the relative amounts of T3R (light stippled bars), RXR (dark stippled bars), and SMRT (black bars) bound to the DR-4 DNA under the different conditions were quantified (C). Although the experiment was quantified in both the absence and presence of T3 hormone, electrophoretograms are provided only for the experiment performed in the absence of T3.

The Preferential Association of SMRT with T3R Homodimers Was Also Observed for a Variety of Natural and Synthetic DNA Response Elements-- We next sought to determine if the preferential binding of SMRT to homodimers of T3R, observed on DR-4s, extended to other classes of DNA response elements. We first compared DR-4, DIV-6, and INV-0 response elements (49). T3Rbeta was able to form homodimers on all of the DNA elements tested, and in all three cases these T3Rbeta homodimer complexes were efficiently supershifted by the addition of GST-SMRT in the absence but not in the presence of 1 µM T3 hormone (Fig. 4A). For all three elements, the addition of RXR led to the formation of a novel complex with properties characteristic of a T3R·RXR heterodimer, and in all cases this T3R·RXR heterodimer was less susceptible to supershift by GST-SMRT than was the T3R·T3R homodimer (Fig. 4A). The DR-4, DIV-6, and INV-0 elements are synthetically derived, artificially optimized response elements; we therefore also extended our studies to two naturally occurring HREs that play a role in T3R function in vivo: the rat growth hormone promoter element and the chicken lysozyme silencer element (cLYS) (48, 62). T3R homodimers formed on these naturally occurring elements were efficiently supershifted by GST-SMRT, whereas the corresponding T3R·RXR heterodimers were not (data not shown). We conclude that for all DNA elements tested, the precise sequence and topology of the response element does not directly affect the ability of the tethered T3R to interact with SMRT corepressor and that the T3R·T3R homodimer displayed a significantly stronger interaction with SMRT than did the T3R·RXR heterodimer.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   Preferential interaction of SMRT with T3Rbeta homodimers as detected on different DNA response elements. A, the ability of SMRT to preferentially interact with T3Rbeta ·T3Rbeta homodimers, relative to T3Rbeta ·RXRalpha heterodimers, was examined by EMSA for a DR-4 element, a DIV-6 element, or an INV-0 element. The EMSAs were performed and quantified as in Fig. 2A. For each DNA element, at least three independent experiments were performed, with the averages and the S.D. values shown. B, T3Rbeta homodimers bound to different response elements exhibit a similar release of corepressor in response to hormone. The EMSA analysis was performed as described in A, except that a range of hormone concentrations from 0 to 1000 nM T3 was used. At each hormone concentration, the amount of T3R·T3R·SMRT complex was determined relative to the amount of total T3R·T3R complex. The amount of T3R·T3R·SMRT complex formed in the absence of T3 is defined as 100% for comparison purposes.

T3Rs Bound to Different Response Elements Exhibited a Similar Release of Corepressor in Response to Hormone-- The experiments detailed above tested the interaction of T3R and T3R·RXR with corepressor in either the absence of hormone or the presence of a large excess (1 µM) of T3 ligand. We wished to determine if binding of these receptor complexes to different response elements might subtly influence the ability of hormone to displace corepressor at intermediate hormone concentrations. We therefore repeated our previous experiments using T3Rbeta , three different response elements, and a range of hormone concentrations from 0.1 to 1000 nM. The results are quantified in Fig. 4B. No significant difference could be detected among the different response elements in the ability of hormone to release SMRT from the T3R homodimer complex. Approximately 12 nM T3 was sufficient to displace 50% of the SMRT complexes from any of the elements tested: the DIV-6, INV-0, and DR-4. We conclude that at the level of resolution of this in vitro experiment, different DNA response elements do not alter the ability of hormone to release corepressor.

Overexpression of RXR in Transfected Cells Counteracts T3R-mediated Repression-- Our results suggested that homodimers, and not heterodimers, of T3R may serve to confer target gene repression in vivo. To test a prediction of this hypothesis, we examined the ability of T3Rs to repress reporter gene expression in transfected cells in the presence of increasing amounts of RXR. CV-1 cells lack endogenous T3Rs, and a DR-4-luciferase reporter displayed a basal level of expression when introduced into these cells (Fig. 5). Co-introduction of a T3Ralpha expression plasmid in the absence of hormone resulted in a reduction in luciferase activity to below basal levels (Fig. 5); this repression reflects the ability of T3R to recruit the SMRT corepressor complex (e.g. Refs. 15 and 20). If the same levels of T3Ralpha were maintained but increasing amounts of an RXR expression construct were introduced into the CV-1 cells, the T3R-mediated repression was counteracted, resulting in partial restoration of basal reporter gene expression (Fig. 5). These results were most clearly observed with the DR-4 and INV-0 elements but were also seen more weakly with a DIV-6 element. These effects of RXR on luciferase activity were not observed in the absence of T3R, were not observed with an otherwise identical reporter construct lacking a hormone response element, and were not due to a reduction in the levels of T3R expressed in the transfected cells (Fig. 5 and data not shown). Therefore, these results, together with our in vitro data, suggest that formation of T3R·RXR heterodimers from T3R·T3R homodimers inhibits the ability of corepressor to interact with the receptor and to mediate transcriptional repression.


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of increasing RXRalpha on T3R-mediated repression. The ability of T3Ralpha to repress reporter gene expression in transfected cells was analyzed in the presence of increasing amounts of an RXRalpha expression vector. CV-1 cells were transfected with 50 ng of either an empty pSG5 vector (None) or a pSG5-T3Ralpha vector, together with 200 ng of a pCH110 lacZ vector and 200 ng of a thymidine kinase-luciferase reporter containing a DR-4, DIV-6, or INV-0 response element, as indicated below. 0 ng (light stippled bars), 10 ng (dark stippled bars), or 50 ng (black bars) of a pSG5-RXRalpha vector were included in each transfection; appropriate amounts of an empty pSG5 vector were used to keep the total amount of vector equal in all cases. The cells were subsequently incubated and harvested, and the luciferase activity was determined and normalized relative to the beta -galactosidase activity. The averages and S.D. values of at least two experiments are presented.

A T3Rbeta Mutant Associated with Human Resistance to Thyroid Hormone Syndrome, Delta 432-T3Rbeta , Exhibits Increased Homodimer Formation, Increased Interaction of the Homodimer with SMRT, and Enhanced Dominant Negative Repression Properties in Transfections-- Mutations in the T3Rbeta locus occur naturally in the human population and can manifest as an endocrine disorder, RTH syndrome. In most cases characterized, the mutant T3Rs associated with RTH syndrome are unable to release properly from corepressor in response to T3 hormone (56, 63-66). Apparently as a consequence, these RTH-T3Rs act as repressors and can interfere in a dominant negative fashion with the functions of the wild-type T3Rs. Intriguingly, several RTH-T3R mutants not only fail to release from corepressor upon the addition of T3 hormone but also display an enhanced corepressor interaction in the absence of hormone (53). We wished to determine if the elevated interaction of these RTH-T3R mutants with SMRT, previously characterized in solution, was also observed when the receptor was bound to DNA and if this enhanced corepressor interaction was specific to the homodimeric form of the receptor.

The Delta 432-T3Rbeta mutant, representing an in frame deletion within helix 11 of the hormone binding domain, displays a 4-8-fold enhanced interaction with SMRT compared with wild-type receptor in a GST pull-down protocol (53). Intriguingly, the Delta 432-T3Rbeta mutant formed homodimers on the DR-4 DNA element somewhat more readily than did the wild-type T3Rbeta (Fig. 6A, compare lane 7 with lane 3), and virtually all of the Delta 432-T3Rbeta homodimer complex was supershifted by SMRT under conditions where less than half of the wild-type T3Rbeta homodimer was supershifted (Fig. 6A, compare lane 9 with lane 5). We conclude that the enhanced ability of the Delta 432-T3Rbeta mutant to interact with SMRT, previously seen in solution, extends to receptor homodimers formed on HREs. We next asked if the enhanced SMRT interaction observed with the Delta 432 mutant applied only to T3R homodimers or was also manifested for heterodimers. Paralleling our results with the wild-type receptor, heterodimers formed by the Delta 432-T3Rbeta mutant in the presence of excess RXR were much less susceptible to supershift by SMRT than were the corresponding Delta 432-T3Rbeta homodimers (Fig. 6A, compare lanes 13 and 14 with lanes 11 and 12). Consistent with these enhanced homodimerization and SMRT association properties in vitro, repression by the Delta 432-T3Rbeta mutant was more resistant to the inhibitory effects of ectopic RXR than was repression by wild-type T3Rs (Fig. 6B), and the Delta 432-T3Rbeta mutant exhibits enhanced dominant negative properties in transfected cells (53). We conclude that the elevated ability of the Delta 432-T3Rbeta mutant to recruit SMRT is largely restricted to homodimers of this receptor and that this enhanced corepressor/homodimer interaction in vitro is paralleled by an augmented ability of the Delta 432 mutant to repress transcription in vivo.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 6.   Enhanced homodimer formation and enhanced corepressor binding by the Delta 432 mutant T3Rbeta . A, SMRT corepressor strongly interacts with Delta 432-T3Rbeta homodimers, but not with RXR·Delta 432-T3Rbeta heterodimers. Wild-type or Delta 432 mutant T3Rbeta receptors, bound to a DR-4 DNA element, were examined for the ability to interact with GST-SMRT. EMSAs were performed as described in the legend to Fig. 1B, except that in vitro transcription/translations were employed as a source of receptor (see "Experimental Procedures"). Two faint, background complexes (denoted by an asterisk) were observed in many lanes, including DNA binding reactions performed with an unprogrammed transcription/translation lysate (lanes 1 and 2). Wild-type T3Rbeta alone, the Delta 432-T3Rbeta mutant alone, or the Delta 432-T3Rbeta mutant together with RXR were tested in the presence or absence of SMRT, as indicated below. B, repression by the Delta 432-T3Rbeta mutant is more resistant to the effects of RXR than is repression by wild-type T3R. The same experimental strategy as in Fig. 5 was employed but testing the Delta 432-T3Rbeta mutant in addition to the wild-type T3Ralpha and -beta alleles, as indicated below.

v-Erb A, an Oncogenic Form of T3Ralpha , Displays an Enhanced Ability to Homodimerize on Certain Response Elements and, as a Consequence, an Enhanced Interaction with SMRT Corepressor-- The v-erb A oncogene was first identified as a locus involved in leukemogenesis by avian erythroblastosis virus and was subsequently recognized to be a virally transduced version of the T3Ralpha gene (7, 67). Compared with the wild-type receptor, v-Erb A bears a C-terminal deletion that prevents hormone binding; as a consequence, v-Erb A interacts with corepressor in both the absence and the presence of hormone and is thought to function in oncogenesis as a dominant negative inhibitor of T3Rs and retinoid receptors (8, 55, 68-70). Notably, v-Erb A has sustained multiple mutations compared with the wild-type T3Ralpha , including 13 amino acid substitutions (71); many of these mutations enhance the oncogenic abilities of the v-Erb A protein and appear to have been selected for during viral propagation (e.g. Refs. 72 and 73). Among other differences between v-Erb A and its T3Ralpha progenitor, it has been reported that v-Erb A is unable to form heterodimers with RXR (74); however, contrary results have also been obtained that suggest v-Erb A does heterodimerize with RXRs, although at reduced efficiency compared with T3Ralpha (55). No role for the altered dimerization properties of v-Erb A has previously been ascertained.

To resolve this apparent conflict in the literature, we determined if v-Erb A preferentially forms homodimers, rather than heterodimers, only on specific DNA elements and, if so, what the consequences might be for corepressor recruitment. We first examined the ability of v-Erb A to bind to the DR-4 and the cLYS (DIV-6) elements. In the absence of RXR, the v-Erb A protein bound to both DNA elements with a mobility characteristic of v-Erb A homodimers; no equivalent complex was observed in control experiments using unprimed reticulocyte lysates (Fig. 7B). Intriguingly, v-Erb A formed homodimers on these DNA probes more readily than did T3Ralpha , with this effect particularly evident for the cLYS element (Fig. 7B, compare lanes 8 with lanes 3). The addition of RXR to the v-Erb A preparation enhanced the ability of the oncoprotein to bind to the DR-4 element but had little or no effect on the already strong ability of v-Erb A to bind to the cLYS element (Fig. 7B). Due to their comparable charge/mass ratios, v-Erb A·RXR heterodimers migrate at virtually the same position in these electrophoretograms as do v-Erb A homodimers (Fig. 7B). Use of receptor-specific antibodies, however, confirmed that the enhanced binding of v-Erb A to the DR-4 element upon the addition of RXR corresponds to the formation of v-Erb A·RXR heterodimers, whereas the bulk of v-Erb A on the cLYS element remains a homodimer and does not associate with RXR under the same conditions (Fig. 8 and data not shown). In contrast, the addition of increasing amounts of RXR to the T3Ralpha preparation resulted in the formation of receptor heterodimers and increased DNA binding on both the DR-4 and cLYS elements (Fig. 7B, lanes 4-7), although heterodimer formation was again somewhat more efficient on the DR-4 (Fig. 7B, compare lanes 4-7 in the upper and lower panels). We conclude that v-Erb A forms homodimers more efficiently than does T3Ralpha , particularly on the cLYS element, and that v-Erb A can associate and form heterodimers with RXR on a DR-4, whereas v-Erb A remains a homodimer and exhibits little or no ability to form heterodimers with RXR on the cLYS element.


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 7.   More efficient formation of homodimers by v-Erb A compared with T3Ralpha . A, schematic representations of the protein structure of avian T3Ralpha (top) and v-Erb A (bottom) are shown. Deletions in v-Erb A compared with chicken T3Ralpha are indicated, as are amino acid substitutions (vertical bars); gag refers to retroviral structural sequences present on the N terminus of the viral protein. B, the ability of v-Erb A to form heterodimers with RXRalpha is compared with that of T3Ralpha . EMSAs were performed using either 6 µl of unprogrammed translation product (lanes 1 and 2), 4 µl of T3Ralpha with increasing amounts of RXRalpha translation product (0, 0.5, 1, or 2 µl; lanes 3-7), or 4 µl of v-erb A with increasing amounts of RXRalpha (0, 0.5, 1, or 2 µl; lanes 8-11). Analyses were performed on both the DR-4 (top panel) and the cLYS (bottom panel) elements in the presence or absence of T3 as indicated. The positions of the v-Erb A homodimer and v-Erb A·RXR heterodimer complexes are indicated to the right (denoted vA/vA and vA/RXR, respectively).


View larger version (79K):
[in this window]
[in a new window]
 
Fig. 8.   Enhanced corepressor recruitment by v-Erb A homodimers. EMSAs were performed as described in the legend to Fig. 7 using either the DR-4 or the cLYS elements and including either 100 ng of GST or GST-SMRT in the binding reactions, as indicated. RXR-directed (X) or normal sera (N) were also included in the reactions as indicated. The locations of protein-DNA complexes corresponding to v-Erb A·v-Erb A homodimers, v-Erb A·v-Erb A homodimers supershifted by SMRT, and v-Erb A·RXR heterodimers supershifted by RXR antibody are indicated to the right (vA/vA, vA/vA/SMRT, and vA/RXR*, respectively).

Are these different dimerization properties of v-Erb A manifested as different interactions with corepressor? In the absence of RXR, v-Erb A homodimers formed on either the DR-4 or the cLYS elements were supershifted by SMRT, with the enhanced formation of v-Erb A homodimers on the cLYS element reflected as an enhanced formation of SMRT complex (Fig. 8, lanes 5 and 6 and lanes 14 and 15). This difference between the ability of SMRT to bind to v-Erb A on the two different response elements was further magnified in the presence of RXR. The addition of RXR to v-Erb A on the DR-4 element resulted in a partial conversion of the v-Erb A homodimer to a v-Erb A·RXR heterodimer complex; the addition of SMRT to this mixed population preferentially supershifted the v-Erb A homodimeric complex but left unaltered the majority of the v-Erb A·RXR heterodimeric complex (Fig. 8, compare lanes 8 and 7). In contrast to the DR-4, the cLYS element, which selected virtually exclusively for v-Erb A homodimers, generated an extremely strong interaction of v-Erb A with SMRT corepressor (Fig. 8, compare lanes 19 and 18). We conclude that v-Erb A displays an elevated ability to form homodimers and a decreased ability to form heterodimers relative to the T3Ralpha progenitor, that this preferential formation of homodimers by v-Erb A is dependent on the nature of the DNA response element, and that enhanced homodimerization properties of v-Erb A are reflected as an enhanced ability of v-Erb A to recruit SMRT corepressor.

RARs, Unlike T3Rs, Interact with SMRT Corepressor both as Receptor Homodimers and as Heterodimers with RXRs-- We examined if the preferential recruitment of SMRT by T3R homodimers extended to other members of the nuclear receptor family. Unliganded RARs, like unliganded T3Rs, interact with SMRT and can form either homodimers or heterodimers with RXR (3, 4, 15, 17, 20). We therefore tested RARs in our EMSA procedure. RARalpha formed receptor homodimers when incubated with a suitable DNA response element (i.e. a DR-5 element) (Fig. 9A). The RARalpha homodimers were efficiently supershifted to a slower electrophoretic mobility by the addition of GST-SMRT, whereas the addition of a hormone agonist (all-trans-retinoic acid; ATRA) dissociated this presumptive RAR·RAR·SMRT complex (Fig. 9A, compare lanes 3 and 4 with lanes 1 and 2). The addition of RXRalpha to the RARalpha preparation resulted in the formation of receptor heterodimers that bound to the DR-5 element with higher affinity than did the RAR homodimers (Fig. 9A, lanes 5-8; only one-eighth of the RARalpha preparation employed in lanes 1-4 was utilized in lanes 5-8). In common with the RARalpha homodimers, but in marked contrast to T3R·RXR heterodimers, the RARalpha ·RXRalpha heterodimers were efficiently supershifted by the addition of GST-SMRT (Fig. 9A, compare lanes 7 and 5). This presumptive RAR· RXR·SMRT complex appeared to be authentic; it was not observed if RXR or SMRT was employed in the EMSA in the absence of RARalpha , the mobility of the RAR·RXR·SMRT complex was further supershifted by RXR-directed antibodies, and the complex was dissociated by the addition of all-trans-retinoic acid (Fig. 9B and data not shown). We conclude that the preference of SMRT for T3R homodimers does not extend to all members of the nuclear receptor family and that the nature of the RARalpha dimer does not significantly alter the ability of this receptor to interact with corepressor under the conditions tested here.


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 9.   Interaction of SMRT both with RAR homodimers and with RAR·RXR heterodimers. A, the ability of SMRT to interact with both RAR homodimers and RAR·RXR heterodimers was analyzed by EMSA. Either RARalpha alone (lanes 1-4) or RARalpha and RXRalpha together (lanes 5-8) were incubated with a 32P-labeled DR-5 DNA element in the presence or absence of all-trans-retinoic acid (ATRA), as indicated below. GST (-) or GST-SMRT (+) was included in the reactions as indicated. The positions of the protein-DNA complexes corresponding to RAR·RAR homodimers, RAR·RXR heterodimers, and the corresponding complexes supershifted with SMRT are indicated to the right. B, RAR·RXR heterodimers interact with SMRT and can be supershifted by RXR antibody. A similar experiment was performed as in A, except that in vitro transcription/translation reactions were used as a source of RAR. RXR-directed antiserum (X) was included in the binding reactions as indicated. The positions of the protein-DNA complexes supershifted by the RXR-directed antiserum are indicated by asterisks.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Efficacy of Corepressor Recruitment by T3Rs Is Contingent on the Nature of the Receptor Dimer-- Our studies demonstrate that the SMRT corepressor is recruited at high affinity to homodimers of T3R assembled on DNA but not to heterodimers composed of T3R and RXR. The strong preference of SMRT for T3R homodimers over heterodimers extended to an assortment of synthetic and natural DNA response elements, was observed for a variety of T3R and RXR isoforms, and was detected by both an EMSA supershift protocol and by an avidin-biotin DNA binding procedure. The selectivity of the SMRT/receptor interaction was most clearly demonstrated in experiments employing a mixed population of T3R homodimers and T3R·RXR heterodimers. The addition of SMRT to this mixed receptor population resulted in a selective interaction of the corepressor with the T3R homodimers; in contrast, little or no direct interaction of SMRT was observed with the T3R·RXR heterodimers simultaneously present in the same receptor population. Intriguingly, the addition of SMRT was able to reduce the abundance of T3R·RXR heterodimers in these experiments without inducing formation of a corresponding T3R·RXR·SMRT complex. Experimental dissection of this phenomenon revealed that the loss of T3R·RXR heterodimers was probably a mass action effect. Presumably, by selectively interacting with T3R homodimers, SMRT reduces the availability of T3R for heterodimerization with RXR, thereby altering the dynamic equilibrium between receptor homodimers and receptor heterodimers. This ability of SMRT to recruit T3R homodimers at the expense of T3R·RXR heterodimers may also play a role in stabilizing the formation of T3R homodimers in vivo (see below).

At first glance, our results might appear to contradict prior studies reporting RXR as stimulating, rather than inhibiting, SMRT recruitment by T3Rs (58, 60). However, most of these previous studies employed GST pull-down and two-hybrid methods to assay the corepressor/receptor interaction, leaving the precise nature of the resulting receptor "dimers" undefined. It is likely that authentic T3R homodimers do not form in GST-pull-down and two-hybrid assays; the effects of RXR in these cases may reflect a preference of SMRT for T3R·RXR heterodimers (which do form in solution) relative to T3R monomers, not relative to homodimers. In support of our own findings with SMRT, N-CoR, a protein that displays 50% sequence relatedness to SMRT, displays a similar preference for T3R homodimers in EMSA experiments (75, 76). In this regard, we (and others) have found that SMRT and N-CoR interact comparably with T3Rs (20, 58, 60); a suggestion to the contrary, that N-CoR interacts with T3Rs more strongly than SMRT, may be the result of differences in the C terminus of the SMRT and N-CoR constructs employed in these prior experiments (76).

Do T3R homodimers mediate transcriptional repression in vivo? In this regard, a number of studies have implicated RXR heterodimers in transcriptional activation, but much less is known as to the nature of the receptor entity involved in transcriptional repression (77, 78). Indeed, several features of T3R molecular biology support a role for T3R homodimers in transcriptional repression. (a) It is difficult to reconcile the poor ability of T3R·RXR heterodimers to interact with SMRT in vitro with the proposal that heterodimers function in repression in vivo. (b) Although T3R homodimers are generally less stable than are T3R·RXR heterodimers, the presence of SMRT can change this equilibrium by stabilizing homodimers and destablizing heterodimers. The levels of SMRT protein in vivo may be sufficient to allow the formation of T3R homodimers on appropriate DNA elements in preference to T3R·RXR heterodimers. (c) Conversely, the addition of T3 hormone can destabilize T3R homodimers but not T3R·RXR heterodimers (43, 52, 61, 79, 80). Thus, binding of hormone may induce release of SMRT by two means: a direct allosteric change in the receptor and a shift in the nature of the receptor-DNA complex from a corepressor-interactive T3R homodimer to a corepressor-noninteractive T3R·RXR heterodimer. (d) Ectopic expression of high levels of RXR in transfected cells counteracts the ability of T3Rs to repress. This RXR-mediated inhibition of repression is opposite to the stimulatory effects of RXR on T3R-mediated activation and suggests that altering the T3R equilibrium from homodimers to heterodimers is paralleled by a shift from repression to activation. (e) DNA response elements and mutants of T3R that favor formation of T3R homodimers also favor receptor-mediated transcriptional repression; this last point is discussed at greater length below.

What Is the Molecular Basis of the Specificity Exhibited by SMRT for T3R Homodimers?-- The ability of receptor dimers to recruit SMRT or N-CoR more efficiently than receptor monomers is well established and presumably relates to the stoichiometry of the receptor-corepressor complex (60). SMRT contains two (L/I)XXII motifs, each of which can interact with a hydrophobic groove on the surface of the nuclear receptor; the corepressor molecule therefore contains two potential receptor interaction sites, whereas only one corepressor interaction site has been mapped on the corresponding surface of the nuclear receptor (33, 35, 36). Receptor dimer formation may, therefore, stabilize the corepressor interaction by permitting occupancy of both of the two receptor interaction sites within a single corepressor molecule. Alternatively the enhanced ability of SMRT to be recruited by receptor dimers may reflect the ability of the corepressor itself to dimerize, thereby allowing a dimeric corepressor/dimeric receptor interaction to occur.

Stoichiometric considerations may account for the preference of SMRT for receptor dimers, but they do not explain why T3R homodimers recruit corepressor better than do T3R·RXR heterodimers. RXR alone, when assayed by GST pull-down or two-hybrid procedures, has a much lower affinity for SMRT than does T3R (9, 15, 18, 20, 21, 58). The presence of two strong SMRT interaction sites in the T3R·T3R homodimer might therefore tether corepressor more strongly than the one strong and one weak interaction sites available in the T3R·RXR heterodimer. However, this hypothesis does not account for the strong SMRT interaction we observe for the RAR·RXR heterodimer, which would be expected to display an analogous mix of one strong and one weak interaction site (RARs, when tested alone, possess a similar affinity for SMRT as do T3Rs). Of note, the low affinity of RXR for corepressor has been attributed to a steric inhibition by the RXR C-terminal helix 12 domain (81). Formation of a RAR·RXR heterodimer might reposition the RXR C terminus so as to alleviate this steric inhibition, resulting in a strong RXR/SMRT interaction, whereas the geometry of the T3R·RXR heterodimer may be unable to alleviate the RXR helix 12-mediated inhibition. Indeed, deletion of the RXR helix 12 modestly enhances the ability of the T3R·RXR heterodimer to recruit SMRT although not to the level observed for the T3R homodimer.2 Other receptor and corepressor determinants may also be involved in this phenomenon; for example, T3Rs interact with both (L/I)XXII interaction domains within SMRT, whereas RARs and RXRs display preferences for individual interaction domains (18, 20). We are currently pursuing experiments to define the contributions of these different interaction surfaces to the homodimer/heterodimer phenomenon.

The v-Erb A Protein Exhibits an Enhanced Ability to Form Homodimers and to Recruit Corepressor, Compared with the T3Ralpha (c-Erb A) Progenitor-- The v-erb A gene was first identified as an oncogenic locus within the avian erythroblastosis virus and was later recognized to be a virally transduced version of the normal avian T3Ralpha transcript (7, 67). v-Erb A has sustained 13 internal amino acid substitutions, as well as N- and C-terminal deletions, compared with the T3Ralpha progenitor, and as a consequence v-Erb A acts as a constitutive repressor of genes normally regulated by T3Rs and by RARs (7, 8, 55, 68). When examined, many of these differences between v-Erb A and the T3Ralpha progenitor contribute to the oncogenic properties of the former, and these v-Erb A mutations may be the result of a selection for increased oncogenic virulence during viral propagation. For example, mutations in the C terminus of v-Erb A result in an inability of the viral protein to release from corepressor in response to hormone, thereby generating the constitutive repressor phenotype (70). Notably, four different mutations, within the v-Erb A DNA binding domain, have changed the DNA recognition properties of v-Erb A, and this alteration in DNA recognition also appears necessary for efficient oncogenesis (55, 72, 73).

v-Erb A has been reported to differ from T3Ralpha in at least one additional aspect: the ability to interact with RXR. It has been reported that v-Erb A forms homodimers but does not heterodimerize with RXR (74). However, alternative reports have been published that indicate v-Erb A can form heterodimers with RXR although at a reduced efficiency relative to T3Ralpha (55). The results obtained in the current study help reconcile these apparently opposing views; v-Erb A is impaired in its ability to form heterodimers with RXR, but the extent of this impairment is highly dependent on the nature of the DNA response element. On a DR-4 element, v-Erb A can form both homodimers and heterodimers with RXR, although the latter occurs with reduced efficacy compared with T3Ralpha . On a DIV-6 element (such as the cLYS promoter sequence) v-Erb A binds very strongly as a homodimer and exhibits little or no ability to form heterodimers with RXR. In this regard, v-Erb A displays, in a highly magnified manner, an otherwise similar preference observed for T3R; the cLYS element recruits T3R homodimers better than does the DR-4 element, and more RXR must be added to generate T3R·RXR heterodimers on the cLYS element than on the DR-4 element. Nonetheless, on all DNA elements we tested here, v-Erb A was significantly more resistant to RXR heterodimer formation than was T3Ralpha , with v-Erb A binding to the cLYS element as a receptor homodimer even at very high RXR concentrations.

Notably, the ability of v-Erb A to bind to the cLYS element very strongly as a homodimer resulted in a very efficient recruitment of SMRT corepressor, and this ability to recruit SMRT was not inhibited by the presence of high levels of RXR. In contrast, v-Erb A homodimers formed on the DR-4 element could be driven into heterodimers by the addition of large amounts of RXR, and these v-Erb A·RXR heterodimers were significantly less able to recruit SMRT corepressor than were the v-Erb A homodimers. We therefore propose that the enhanced homodimerization and impaired heterodimerization properties of the v-Erb A protein are likely to contribute to the ability of the viral protein to recruit corepressor and to act as a transcriptional repressor in cells where RXR is present. The enhanced homodimerization properties of v-Erb A may therefore operate together with the defects in hormone binding and altered DNA recognition properties, noted previously, to enhance the leukemogenic proclivities of the v-Erb A oncoprotein by making it a stronger antimorph. The extent to which this enhanced homodimerization phenomenon is manifested is dependent on the nature of the DNA binding site and therefore is likely to differ for different promoters. Of note, the T3Rbeta Delta 432 mutation implicated in human RTH syndrome also displays an increased ability to form homodimers, an enhanced interaction with SMRT, and an enhanced ability to function as a dominant negative repressor. Therefore, mutations that increase the ability of T3Rs to homodimerize may have the general effect of increasing the ability of the receptor to tether SMRT and to function as a transcriptional repressor.

The Nature of the DNA Response Element and the Nature of the Receptor Dimer Determine Corepressor Recruitment-- The T3Rs are unique among the nuclear hormone receptors in their ability to bind to response elements exhibiting an extraordinary range of different half-site spacing and orientations. Different thyroid hormone response elements can mediate different transcriptional regulatory properties. For example, DIV-6 sequences such as the cLYS element mediate strong transcriptional repression and relatively weak transcriptional activation by T3Rs, whereas DR-4 elements tend to exhibit the reciprocal properties (48, 52, 82). We therefore examined if the different topological organizations of these different elements could influence the ability of the tethered T3Rs to bind or release from corepressor. When using pure T3R homodimers, the orientations of the half-sites did not appear to directly influence corepressor recruitment; all three DNA elements tested (DR-4, INV-0, and DIV-6) exhibited comparable corepressor association and hormone-mediated dissociation. Notably, however, these elements do differ in the ability to recruit T3Rs as homodimers or as heterodimers with RXR, and, as a consequence, corepressor recruitment by the different elements differed in the presence of a mix of T3Rs and RXRs. Most obvious was the enhanced homodimerization and impaired heterodimerization properties of v-Erb A on the cLYS element, which represents a DIV-6 half-site orientation. Interestingly, the cLYS element was initially isolated from avian erythroblastosis virus-transformed erythroid cells by virtue of its ability to confer v-Erb A-mediated repression of lysozyme expression (48). Native T3Ralpha also formed homodimers more readily (and heterodimers less readily) on the cLYS element than on a DR-4 element, although this phenomenon was more muted than for v-Erb A. We suggest that the ability of different hormone response elements to exhibit different transcriptional properties may reflect, in part, differences in the ability of these DNA sequences to recruit receptor homodimers versus heterodimers, which in turn can influence the interactions of these receptor complexes with corepressors.

Presenting an interesting contrast to this phenomenon for T3Rs, RARalpha interacts strongly with SMRT corepressor whether tested as a receptor homodimer or as a heterodimer with RXR. Presumably, the nature of the receptor dimer does not play as critical a role in determining RAR-mediated transcriptional regulation as it does for the T3Rs; however, an explanation of the physiological or evolutionary basis behind this phenomenon remains elusive. It may be relevant in this regard that retinoic acid does not destabilize the binding of RAR homodimers to DNA, and thus, unlike T3Rs, hormone would not be expected to potentiate an exchange of RAR·RXR heterodimers for RAR homodimers. Nonetheless, it should be noted that an oncogenic fusion protein, PML-RARalpha , derived by chromosomal translocation of the normal RARalpha locus, exhibits an enhanced ability to form homodimers (and homo-oligomers) compared with the unmodified RARalpha , and this enhanced homodimer formation appears to manifest as an enhanced ability of PML-RARalpha to bind to corepressor (83, 84). Although we have not observed differences in the corepressor interaction properties of homo- and heterodimers of native RAR, our experiments do not exclude the possibility that aberrant RAR homodimers, formed by chromosomal rearrangements or by artificial means, may exhibit the enhanced corepressor interactions that we report here for T3R homodimers.

    ACKNOWLEDGEMENT

We thank Valentina Taryanik for dedicated technical assistance.

    FOOTNOTES

* This work was supported by Public Health Services/National Institutes of Health Grants R37 CA-53394, R01 DK-53528, and RO1 DK-54064.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.

Dagger To whom correspondence should be addressed. Tel.: 530-752-3013; Fax: 530-752-9014; E-mail: mlprivalsky{at}ucdavis.edu.

Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M010022200

2 S. M. Yoh and M. L. Privalsky, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: HRE, hormone response element; T3R, thyroid hormone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; RTH, resistance to thyroid hormone; EMSA, electrophoretic mobility shift assay; cLYS, chicken lysozyme silencer element.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Apriletti, J. W., Ribeiro, R. C., Wagner, R. L., Feng, W., Webb, P., Kushner, P. J., West, B. L., Nilsson, S., Scanlan, T. S., Fletterick, R. J., and Baxter, J. D. (1998) Clin. Exp. Pharmacol. Physiol. 25 (suppl.), 2-11
2. Lazar, M. A. (1993) Endocr. Rev. 14, 184-193[Medline] [Order article via Infotrieve]
3. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schütz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83, 835-839[Medline] [Order article via Infotrieve]
4. Mangelsdorf, D. J., and Evans, R. M. (1995) Cell 83, 841-850[Medline] [Order article via Infotrieve]
5. Meier, C. A. (1997) J. Recept. Signal Transduct. Res. 17, 319-335[Medline] [Order article via Infotrieve]
6. Tsai, M. J., and O'Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486[CrossRef][Medline] [Order article via Infotrieve]
7. Damm, K., Thompson, C. C., and Evans, R. M. (1989) Nature 339, 593-597[CrossRef][Medline] [Order article via Infotrieve]
8. Sap, J., Muñoz, A., Schmitt, J., Stunnenberg, H., and Vennström, B. (1989) Nature 340, 242-244[CrossRef][Medline] [Order article via Infotrieve]
9. Schulman, I. G., Juguilon, H., and Evans, R. M. (1996) Mol. Cell. Biol. 16, 3807-3813[Abstract]
10. Chen, J. D., and Li, H. (1998) Crit. Rev. Eukaryotic Gene Expression 8, 169-190[Medline] [Order article via Infotrieve]
11. Horwitz, K. B., Jackson, T. A., Bain, D. L., Richer, J. K., Takimoto, G. S., and Tung, L. (1996) Mol. Endocrinol. 10, 1167-1177[Abstract]
12. Lin, R. J., Kao, H. Y., Ordentlich, P., and Evans, R. M. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 577-585[Medline] [Order article via Infotrieve]
13. Torchia, J., Glass, C., and Rosenfeld, M. G. (1998) Curr. Opin. Cell Biol. 10, 373-383[CrossRef][Medline] [Order article via Infotrieve]
14. Xu, L., Glass, C. K., and Rosenfeld, M. G. (1999) Curr. Opin. Genet. Dev. 9, 140-147[CrossRef][Medline] [Order article via Infotrieve]
15. Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-457[CrossRef][Medline] [Order article via Infotrieve]
16. Chen, J. D., Umesono, K., and Evans, R. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7567-7571[Abstract/Free Full Text]
17. Hörlein, A. J., Näär, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Söderström, M., Glass, C. K., and Rosenfeld, M. G. (1995) Nature 377, 397-404[CrossRef][Medline] [Order article via Infotrieve]
18. Li, H., Leo, C., Schroen, D. J., and Chen, J. D. (1997) Mol. Endocrinol. 11, 2025-2037[Abstract/Free Full Text]
19. Park, E. J., Schroen, D. J., Yang, M., Li, H., Li, L., and Chen, J. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3519-3524[Abstract/Free Full Text]
20. Sande, S., and Privalsky, M. L. (1996) Mol. Endocrinol. 10, 813-825[Abstract]
21. Seol, W., Mahon, M. J., Lee, Y. K., and Moore, D. D. (1996) Mol. Endocrinol. 10, 1646-1655[Abstract]
22. Zamir, I., Harding, H. P., Atkins, G. B., Hörlein, A., Glass, C. K., Rosenfeld, M. G., and Lazar, M. A. (1996) Mol. Cell. Biol. 16, 5458-5465[Abstract]
23. Ayer, D. E. (1999) Trends Cell Biol. 9, 193-198[CrossRef][Medline] [Order article via Infotrieve]
24. Guenther, M. G., Lane, W. S., Fischle, W., Verdin, E., Lazar, M. A., and Shiekhattar, R. (2000) Genes Dev. 14, 1048-1057[Abstract/Free Full Text]
25. Hassig, C. A., and Schreiber, S. L. (1997) Curr. Opin. Chem. Biol. 1, 300-308[CrossRef][Medline] [Order article via Infotrieve]
26. Huang, E. Y., Zhang, J. S., Miska, E. A., Guenther, M. G., Kouzarides, T., and Lazar, M. A. (2000) Genes Dev. 14, 45-54[Abstract/Free Full Text]
27. Kao, H. Y., Downes, M., Ordentlich, P., and Evans, R. M. (2000) Genes Dev. 14, 55-66[Abstract/Free Full Text]
28. Laherty, C. D., Billin, A. N., Lavinsky, R. M., Yochum, G. S., Bush, A. C., Sun, J. M., Mullen, T. M., Davie, J. R., Rose, D. W., Glass, C. K., Rosenfeld, M. G., Ayer, D. E., and Eisenman, R. N. (1998) Mol. Cell. 2, 33-42[Medline] [Order article via Infotrieve]
29. Li, J. W., Wang, J., Wang, J. X., Nawaz, Z., Liu, J. M., Qin, J., and Wong, J. M. (2000) EMBO J. 19, 4342-4350[Abstract/Free Full Text]
30. Nomura, T., Khan, M. M., Kaul, S. C., Dong, H. D., Wadhwa, R., Colmenares, C., Kohno, I., and Ishii, S. (1999) Genes Dev. 13, 412-423[Abstract/Free Full Text]
31. Pazin, M. J., and Kadonaga, J. T. (1997) Cell 89, 325-328[Medline] [Order article via Infotrieve]
32. Wolffe, A. P. (1997) Nature 387, 16-17[CrossRef][Medline] [Order article via Infotrieve]
33. Hu, X., and Lazar, M. A. (1999) Nature 402, 93-96[CrossRef][Medline] [Order article via Infotrieve]
34. Lin, B. C., Hong, S. H., Krig, S., Yoh, S. M., and Privalsky, M. L. (1997) Mol. Cell. Biol. 17, 6131-6138[Abstract]
35. Nagy, L., Kao, H. Y., Love, J. D., Li, C., Banayo, E., Gooch, J. T., Krishna, V., Chatterjee, K., Evans, R. M., and Schwabe, J. W. R. (1999) Genes Dev. 13, 3209-3216[Abstract/Free Full Text]
36. Perissi, V., Staszewski, L. M., McInerney, E. M., Kurokawa, R., Krones, A., Rose, D. W., Lambert, M. H., Milburn, M. V., Glass, C. K., and Rosenfeld, M. G. (1999) Genes Dev. 13, 3198-3208[Abstract/Free Full Text]
37. Ito, M., Yuan, C. X., Malik, S., Gu, W., Fondell, J. D., Yamamura, S., Fu, Z. Y., Zhang, X., Qin, J., and Roeder, R. G. (1999) Mol. Cell. 3, 361-370[Medline] [Order article via Infotrieve]
38. Rachez, C., Lemon, B. D., Suldan, Z., Bromleigh, V., Gamble, M., Näär, A. M., Erdjument-Bromage, H., Tempst, P., and Freedman, L. P. (1999) Nature 398, 824-828[CrossRef][Medline] [Order article via Infotrieve]
39. Muscat, G. E., Burke, L. J., and Downes, M. (1998) Nucleic Acids Res. 26, 2899-2907[Abstract/Free Full Text]
40. Wong, C. W., and Privalsky, M. L. (1998) Mol. Cell. Biol. 8, 5500-5510
41. Workman, J. L., and Kingston, R. E. (1998) Annu. Rev. Biochem. 67, 545-579[CrossRef][Medline] [Order article via Infotrieve]
42. Wu, C. (1997) J. Biol. Chem. 272, 28171-28174[Free Full Text]
43. Forman, B. M., and Samuels, H. H. (1990) New Biol. 2, 587-594[Medline] [Order article via Infotrieve]
44. Laudet, V., and Stehelin, D. (1992) Curr. Biol. 2, 293-295
45. Glass, C. K. (1996) J. Endocrinol. 150, 349-357[Abstract/Free Full Text]
46. Naar, A. M., Boutin, J. M., Lipkin, S. M., Yu, V. C., Holloway, J. M., Glass, C. K., and Rosenfeld, M. G. (1991) Cell 65, 1267-1279[Medline] [Order article via Infotrieve]
47. Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) Cell 65, 1255-1266[Medline] [Order article via Infotrieve]
48. Baniahmad, A., Steiner, C., Kohne, A. C., and Renkawitz, R. (1990) Cell 61, 505-514[Medline] [Order article via Infotrieve]
49. Forman, B. M., Casanova, J., Raaka, B. M., Ghysdael, J., and Samuels, H. H. (1992) Mol. Endocrinol. 6, 429-442[Abstract]
50. Kurokawa, R., Yu, V. C., Naar, A., Kyakumoto, S., Han, Z. H., Silverman, S., Rosenfeld, M. G., and Glass, C. K. (1993) Genes Dev. 7, 1423-1435[Abstract]
51. Perlmann, T., Rangarajan, P. N., Umesono, K., and Evans, R. M. (1993) Genes Dev. 7, 1411-1422[Abstract]
52. Piedrafita, F. J., Bendik, I., Ortiz, M. A., and Pfahl, M. (1995) Mol. Endocrinol. 9, 563-578[Abstract]
53. Yoh, S. M., and Privalsky, M. L. (2000) Mol. Cell. Endocrinol. 159, 109-124[CrossRef][Medline] [Order article via Infotrieve]
54. Tzagarakis-Foster, C., and Privalsky, M. L. (1998) J. Biol. Chem. 273, 10926-10932[Abstract/Free Full Text]
55. Chen, H. W., and Privalsky, M. L. (1993) Mol. Cell. Biol. 13, 5970-5980[Abstract]
56. Yoh, S. M., Chatterjee, V. K., and Privalsky, M. L. (1997) Mol. Endocrinol. 11, 470-480[Abstract/Free Full Text]
57. Hong, S. H., and Privalsky, M. L. (2000) Mol. Cell. Biol. 20, 6612-6625[Abstract/Free Full Text]
58. Wong, C. W., and Privalsky, M. L. (1998) Mol. Cell. Biol. 18, 5724-5733[Abstract/Free Full Text]
59. Zhang, J., Zamir, I., and Lazar, M. A. (1997) Mol. Cell. Biol. 17, 6887-6897[Abstract]
60. Zamir, I., Zhang, J., and Lazar, M. A. (1997) Genes Dev. 11, 835-846[Abstract]
61. Yen, P. M., Darling, D. S., Carter, R. L., Forgione, M., Umeda, P. K., and Chin, W. W. (1992) J. Biol. Chem. 267, 3565-3568[Abstract/Free Full Text]
62. Williams, G. R., Harney, J. W., Forman, B. M., Samuels, H. H., and Brent, G. A. (1991) J. Biol. Chem. 266, 19636-19644[Abstract/Free Full Text]
63. CliftonBligh, R. J., deZegher, F., Wagner, R. L., Collingwood, T. N., Francois, I., VanHelvoirt, M., Fletterick, R. J., and Chatterjee, V. K. K. (1998) Mol. Endocrinol. 12, 609-621[Abstract/Free Full Text]
64. Nagaya, T., Fujieda, M., and Seo, H. (1998) Biochem. Biophys. Res. Commun. 247, 620-623[CrossRef][Medline] [Order article via Infotrieve]
65. Safer, J. D., Cohen, R. N., Hollenberg, A. N., and Wondisford, F. E. (1998) J. Biol. Chem. 273, 30175-30182[Abstract/Free Full Text]
66. Tagami, T., and Jameson, J. L. (1998) Endocrinology 139, 640-650[Abstract/Free Full Text]
67. Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruol, D. J., and Evans, R. M. (1986) Nature 324, 641-646[Medline] [Order article via Infotrieve]
68. Sande, S., Sharif, M., Chen, H. W., and Privalsky, M. (1993) J. Virol. 67, 1067-1074[Abstract]
69. Sharif, M., and Privalsky, M. L. (1991) Cell 66, 885-893[Medline] [Order article via Infotrieve]
70. Zenke, M., Muñoz, A., Sap, J., Vennström, B., and Beug, H. (1990) Cell 61, 1035-1049[Medline] [Order article via Infotrieve]
71. Sap, J., Muñoz, A., Damm, K., Goldberg, Y., Ghysdael, J., Leutz, A., Beug, H., and Vennström, B. (1986) Nature 324, 635-640[Medline] [Order article via Infotrieve]
72. Bonde, B. G., Sharif, M., and Privalsky, M. L. (1991) J. Virol. 65, 2037-2046[Medline] [Order article via Infotrieve]
73. Chen, H. W., Smit-McBride, Z., Lewis, S., Sharif, M., and Privalsky, M. L. (1993) Mol. Cell. Biol. 13, 2366-2376[Abstract]
74. Barettino, D., Bugge, T. H., Bartunek, P., Ruiz, M., Sonntagbuck, V., Beug, H., Zenke, M., and Stunnenberg, H. G. (1993) EMBO J. 12, 1343-1354[Abstract]
75. Cohen, R. N., Wondisford, F. E., and Hollenberg, A. N. (1998) Mol. Endocrinol. 12, 1567-1581[Abstract/Free Full Text]
76. Cohen, R. N., Putney, A., Wondisford, F. E., and Hollenberg, A. N. (2000) Mol. Endocrinol. 14, 900-914[Abstract/Free Full Text]
77. Kastner, P., Grondona, J. M., Mark, M., Gansmuller, A., Lemeur, M., Decimo, D., Vonesch, J. L., Dolle, P., and Chambon, P. (1994) Cell 78, 987-1003[Medline] [Order article via Infotrieve]
78. Wan, Y. J. Y., An, D. S., Cai, Y., Repa, J. J., Chen, T. H. P., Flores, M., Postic, C., Magnuson, M. A., Chen, J., Chien, K. R., French, S., Mangelsdorf, D. J., and Sucov, H. M. (2000) Mol. Cell. Biol. 20, 4436-4444[Abstract/Free Full Text]
79. Andersson, M. L., Nordstrom, K., Demczuk, S., Harbers, M., and Vennström, B. (1992) Nucleic Acids Res. 20, 4803-4810[Abstract]
80. Ribeiro, R. C., Kushner, P. J., Apriletti, J. W., West, B. L., and Baxter, J. D. (1992) Mol. Endocrinol. 6, 1142-1152[Abstract]
81. Zhang, J., Hu, X., and Lazar, M. A. (1999) Mol. Cell. Biol. 19, 6448-6457[Abstract/Free Full Text]
82. Williams, G. R., Zavacki, A. M., Harney, J. W., and Brent, G. A. (1994) Endocrinology 134, 1888-1896[Abstract]
83. Lin, R. J., and Evans, R. M. (2000) Mol. Cell. 5, 821-830[CrossRef][Medline] [Order article via Infotrieve]
84. Minucci, S., Maccarana, M., Cioce, M., De Luca, P., Gelmetti, V., Segalla, S., Di Croce, L., Giavara, S., Matteucci, C., Gobbi, A., Bianchini, A., Colombo, E., Schiavoni, I., Badaracco, G., Hu, X., Lazar, M. A., Landsberger, N., Nervi, C., and Pelicci, P. G. (2000) Mol. Cell. 5, 811-820[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.