Dynamic Inhibition of Nuclear Receptor Activation by Corepressor Binding
Young-Chang Sohn1,
Seung-Whan Kim1,
Seunghee Lee1,
Young-Yun Kong,
Doe Sun Na,
Soo-Kyung Lee and
Jae Woon Lee
Department of Life Science (Y.-C.S., S.-W.K., S.L., Y.-Y.K., J.W.L.), Pohang University of Science and Technology, Pohang 790-784, Korea; Faculty of Marine Bioscience & Technology (Y.-C.S.), Kangnung National University, Kangnung 210-702, Korea; Department of Biochemistry (D.S.N.), College of Medicine, University of Ulsan, Seoul 138-736, Korea; and Gene Expression Laboratory (S.-K.L.), The Salk Institute for Biological Studies, San Diego, California 92037
Address all correspondence and requests for reprints to: Jae Woon Lee, Ph.D., Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea. E-mail: jaewoon{at}postech.ac.kr; or Young-Chang Sohn, Ph.D., Faculty of Marine Bioscience & Technology, Kangnung National University, Kangnung 210-702, Korea. E-mail: ycsohn{at}kangnung.ac.kr.
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ABSTRACT
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Nuclear receptors adopt dramatically different conformations in the presence or absence of ligand, and such liganded (holo) and unliganded (apo) receptors are specifically recognized by transcriptional coactivators and corepressors, respectively. These two states likely exist in dynamic equilibrium, contrary to the conventional model of static off and on conformations. First, corepressor SMRT [for silencing mediator of thyroid hormone receptor (TR) and retinoic acid receptor (RAR)] inhibits the interaction of coactivator steroid receptor coactivator-1 with liganded TR/RAR. Second, SMRT enables receptors to adopt apo-form even in the presence of ligand, as demonstrated with limited proteolyses and decreased binding of radiolabeled retinoid to RAR. Finally, chromatin immunoprecipitation results indicate that SMRT and steroid receptor coactivator-1 dynamically compete for receptor bindings in vivo in the presence of ligand. These results suggest that corepressor binding can drive receptors to adopt the apo-state, even in the presence of ligand, and inhibit activated liganded (holo) nuclear receptors in vivo.
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INTRODUCTION
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NUCLEAR RECEPTORS, key regulators of cell growth and differentiation, homeostasis, and development, represent a large family of ligand-dependent transcription factors (reviewed in Ref. 1). The C-terminal ligand binding domain of these proteins harbors an essential ligand-dependent transactivation function, activation function-2, whereas the N terminus of many nuclear receptors includes ligand-independent transactivation function, activation function-1. In the absence of ligand, many nuclear receptors repress transcription of target genes via recruitment of the corepressors, silencing mediator of retinoid and thyroid hormone receptors (SMRT) and nuclear receptor corepressor (N-CoR). These corepressors in turn recruit various histone deacetylases (HDACs) resulting in an inactive chromatin state (reviewed in Refs. 2, 3, 4). Hormone binding triggers the release of corepressors and the subsequent association of an array of coactivators, including steroid receptor coactivator-1 (SRC-1), cAMP response element binding protein-binding protein (CBP)/p300 and activating signal cointegrator-2 (ASC-2) (2, 3, 4). A distinctive structural feature of SRC-1 and related coactivators is the presence of multiple LXXLL motifs referred to as NR boxes (5, 6). The activation function-2 core region (helix 12) was recently shown to undergo a major restructuring upon ligand binding, forming part of a charged clamp that accommodates SRC-1 within a hydrophobic cleft of the receptors ligand binding domain, through direct contacts with these LXXLL motifs (7, 8). Interestingly, the N-CoR/SMRT nuclear receptor interaction motifs share the consensus sequence LXXI/HIXXXI/L (9, 10, 11), representing an extended helix compared with the coactivator LXXLL helix, which is thought to interact with specific residues in the same receptor pocket required for coactivator binding. Corepressor binding results in displacement of the C-terminal helix 12 (12), an essential component of the activation function-2 charge clamp, which allows the longer corepressor helix to interact with the underlying hydrophobic surface. The binding of ligand stabilizes the activated liganded (holo) receptor structure, and recent results demonstrate that corepressor binding also stabilizes the unliganded (apo) receptor structure (12).
The traditional view is that activated receptors such as retinoic acid receptor (RAR) or thyroid hormone receptor (TR) bind to response elements in the promoters of a target gene and remain associated for as long as cognate ligand is present. However, this is inconsistent with a report that the association of estrogen receptor and the SRC-1 family member ACTR is a transient process that is disrupted by direct acetylation of ACTR by CBP/p300 (13). Furthermore, it was recently demonstrated that estrogen receptor and a number of coactivators rapidly associate with estrogen responsive promoters after estrogen treatment, but then dissociate in a cyclic fashion, with cycles of estrogen receptor complex assembly followed by transcription (14). From these studies, we hypothesized that nuclear receptors, even in the presence of ligand, may exist in a dynamic, reversible equilibrium between apo- and holo-states, and that specific coregulatory proteins may modulate this equilibrium.
We demonstrate in this report that corepressor binding can indeed influence receptors to adopt the apo-state, even in the presence of ligand. Thus, the ratio of coactivator to corepressor in a given cell type may determine the potency of transcriptional activation in equilibrium and controlled expression of corepressors/coactivators may represent novel determinants in the combinatorial regulatory circuit of gene transcription in vivo.
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RESULTS AND DISCUSSION
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Corepressor Bindings Actively Inhibit the Interactions of Coactivator and Liganded Receptors
We hypothesized that nuclear receptors, even in the presence of ligand, may exist in a dynamic, reversible equilibrium between apo- and holo-states, and that specific coregulatory proteins may modulate this equilibrium. In agreement with this hypothesis, the 9-cis-RA-dependent interaction between Gal4/SRC-C (a Gal4 fusion protein to SRC-1 residues 568779 containing the receptor-interacting LXXLL motifs) and VP16/RAR in CV-1 cells was reduced by coexpression of either full-length SMRT or SMRT-D (SMRT residues 10601495 containing the receptor-interacting LXXI/HIXXXI/L motifs; Fig. 1A
). Similar results were also obtained with VP16/TR (data not shown). Consistent with these results, the ligand-dependent interactions between 35S-labeled TR and a fusion protein of glutathione-S-transferase (GST) and SRC-C were attenuated by increasing amounts of bacterially expressed and purified SMRT-D but not BSA (Fig. 1B
). These results suggest that corepressor binding may drive a dynamic receptor conformational equilibrium toward the apo-state even in the presence of ligand.

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Figure 1. SMRT Bindings Block the Receptor-SRC-1 Bindings
A, The mammalian two-hybrid tests were executed in CV-1 cells. Open and closed boxes indicate the absence and presence of 0.1 µM of 9-cis-RA, respectively. B, GST pull-down assays were performed with radiolabeled TRß, as previously described (27 ). His-tagged SMRT-D (His-SMRT-D) fragment was bacterially expressed and purified to homogeneity over nickel column. Increasing amounts of BSA or purified His-SMRT-D (0.07, 0.2, and 0.7 µg, respectively) were added to the reactions. Approximately 30% of the total reaction was loaded as input. - and +, Absence and presence of 0.1 µM of T3, respectively.
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Corepressor Bindings Drive Receptors to Adopt the apo-State Even in the Presence of Ligand
To directly test the prediction that corepressor binding actively drives a dynamic receptor conformational equilibrium toward the apo-state even in the presence of ligand, 35S-labeled TR was subjected to limited proteolysis in the presence or absence of T3 and SMRT-D. As previously noted (15), trypsin digestion (10 µg/ml) unraveled two protected fragments of approximately 31 and 28 kDa only in the presence of T3. Formation of these two bands significantly decreased with increasing amounts of SMRT-D but not BSA (Fig. 2A
). Similar results were also obtained with RAR (data not shown). Corroborating these results, addition of SMRT-D, but neither BSA nor SRC-C, dramatically decreased binding of [3H]-labeled 9-cis-RA to GST/RAR (Fig. 2B
). Furthermore, SRC-C at least partially abrogated the effect of SMRT-D on the ligand binding to RAR. To our knowledge, this is the first direct demonstration that corepressor binding can actively interfere with ligand binding. Overall, these results imply that, in the presence of ligand, corepressor and coactivator compete with each other to occupy nuclear receptors, with corepressor binding trapping nuclear receptors in the apo-form, although the exact order of events are currently unknown.

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Figure 2. SMRT Bindings Revert Holo-Receptor to apo-Form
A, 0.1 µM of T3, and increasing amounts of BSA or His-SMRT-D (0.1, 0.3, and 1.0 µg, respectively) were added as indicated. The digestions were performed for 10 min at room temperature with 10 µg/ml of trypsin (15 ). Approximately 40% of the total reaction was loaded as input. B, GST/RAR bound to GST column was incubated with 10 nM of [3H]9-cis-RA either in the absence or presence of the indicated amount of BSA, His-SMRT-D or His-SRC-C, extensively washed, and counted for radioactivity.
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Dynamic Competition between Corepressors and Coactivators to Bind Receptors in Vivo
The above results indicate that the maximum level of ligand-dependent transactivation achievable within a given cell type may depend on level of corepressor expression. Indeed, cotransfection of additional SMRT or SMRT-D expression vector significantly repressed the ligand-dependent transactivation by TR and RAR in a dose-dependent manner (Fig. 3A
and data not shown). In addition, treatment of cells with the histone deacetylase inhibitor trichostatin A (TSA) increased the T3-dependent transactivation by TR, consistent with the idea that TSA decreased the active inhibitory effect of the apo-TR/corepressor complexes formed in the presence of ligand. Alternatively, histone deacetylases enzymatic activities could play a direct role in the corepressors ability to reverse the equilibrium. To characterize the possible in vivo recruitment of SMRT to the retinoid-response element from the RARß2 promoter (ß-RARE), chromatin immunoprecipitation, and semiquantitative PCR were employed (14). Surprisingly, the promoter of the cotransfected ß-RARE-luciferase (Luc) reporter construct was occupied by SMRT even in the presence of ligand (Fig. 3B
). However, the promoter occupancy by SMRT decreased with increasing amounts of cotransfected SRC-1 (compare lanes 1, 3, and 4 in Fig. 3B
) and increased again by additional cotransfected SMRT (compare lanes 4 and 5 in Fig. 3B
). Similar results were also obtained with the endogenous RARß2 promoter except that even higher promoter occupancy by SMRT was obtained with the presence of ligand (data not shown). Overall, these results clearly demonstrate that corepressors, even in the presence of ligand, dynamically compete in vivo with coactivators for binding receptors.

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Figure 3. Dynamic Interplay between SMRT and SRC-1
A, Cotransfection experiments were executed with T3-responsive T3RE-Luc reporter construct. T3 and TSA (0.1 µM) were used where indicated. B, 293T cells were transfected with SMRT or SRC-1 expression vector and treated with 9-cis-RA for 12 h, as indicated. Soluble chromatin from these cells was prepared and immunoprecipitated with monoclonal antibody against SMRT, as recently described (14 ). The final DNA extractions were amplified using pairs of primers that cover the ß-RARE promoter region. The experiments were repeated at least three times and highly reproducible.
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The CoRNR Box Alone Is Not Sufficient to Drive Liganded Receptors to Adopt the apo-State
The CoRNR boxes in SMRT are likely involved with the ability of SMRT to drive liganded receptors to adopt the apo-state. To directly test this possibility, we employed the previously defined SMRT mutants (16). Surprisingly, SMRT-Dm in which the critical hydrophobic residues of two CoRNR motifs were mutated to alanines (Fig. 4A
) was still able to interfere with the retinoid-dependent mammalian two hybrid interactions between Gal4-SRC-C and VP16-RAR (Fig. 4B
). However, SMRT-Dm
S, which is identical to SMRT-Dm except the deletion of the C-terminal motif termed LSD (for lysine-serine-aspartic acid) known to interact with a novel corepressor protein SHARP (for SMRT/HDAC1-associated repressor protein; Ref. 17), was unable to block these interactions (Fig. 4B
). It is important to note that SHARP contains several CoRNR motifs and accordingly associates with RAR only in the absence of ligand (17). Taken together, these results suggest that the CoRNR motifs present in either SMRT or SHARP are likely responsible for the SMRT-mediated impairment of the interactions between liganded RAR and SRC-1 in vivo. Based on these results, we tested whether the presence of CoRNR motifs alone is sufficient to effect the equilibrium toward the apo-state. A chimeric coactivator subGRIP1 (Fig. 4A
), in which all three of the GRIP1 NR boxes are replaced by the CoRNR motifs of SMRT/N-CoR, is known to bind apo-receptors but not liganded receptors (14). In cotransfections, however, subGRIP1 was not able to suppress the ligand-dependent interactions between Gal4/SRC-C and VP16/RAR (Fig. 4B
). It is noted that the expression levels for SMRT-D, SMRT-Dm, SMRT-D
S, and SMRT-Dm
S were comparable with each other (Fig. 4C
). In addition, Western blot analysis with anti-GRIP1 antibody that detects both the wild type GRIP1 and subGRIP1 revealed that GRIP1 immunoreactivity is significantly increased in cells transfected with subGRIP1, as reported (14). Thus, the CoRNR boxes alone may not be sufficient for the corepressor SMRT-mediated reversal of the equilibrium to the apo-state in the presence of ligand. Importantly, these results also demonstrate that SMRT directly regulates the equilibrium by utilizing other functional domains yet to define in addition to the CoRNR boxes, rather than indirectly trapping the apo-receptor after the spontaneous dissociation of ligand from the receptor (18).

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Figure 4. Insufficiency of the CoRNR Boxes to Reverse the Equilibrium
A, Schematic representation of mutant coregulators are as shown (16 ). The NR boxes of the coactivator GRIP1 were mutated to CoRNR sequences in subGRIP1 (14 ). B, The mammalian two-hybrid tests were executed with 50 ng of Gal4/SRC-C and 50 ng of VP16/RAR expression vectors in CV-1 cells, along with expression vectors for SMRT-D, SMRT-Dm, SMRT-D S, SMRT-Dm S, and subGRIP1. The open and closed boxes indicate the absence and presence of 0.1 µM of 9-cis-RA, respectively. C, Western analyses with HA antibody confirmed the comparable expression of SMRT-D, SMRT-Dm, SMRT-D S, and SMRT-Dm S in these transfected cells (B). The expression of subGRIP1 was also confirmed with GRIP1 antibody.
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Inability of Coactivators Alone to Affect the Equilibrium
We tested whether coactivators may similarly drive the equilibrium toward the holo-state in the absence of ligand. In this case, coexpression of coactivators should stimulate the basal level of transactivation by nuclear receptors. However, cotransfection of increasing amounts of SRC-C, the full-length SRC-1, xSRC-3, or ASC-2 expression vector was unable to enhance the basal level of transactivation by either apo-TR or apo-RAR (Fig. 5A
and data not shown). Similarly, the interactions of radiolabeled TR or RAR with GST/SMRT-D were not affected by increasing amounts of SRC-C in the absence of ligand (data not shown). These results strongly suggest that the major force driving the equilibrium to the holo-state is ligand binding but not coactivator binding.

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Figure 5. Inability of Coactivator Alone to Affect the Equilibrium
A, Cotransfection experiments were executed with T3-responsive T3RE-Luc reporter construct for SRC-C, xSRC-3 (29 ), and ASC-2 (26 ). Similar results were also obtained with the full-length SRC-1 (data not shown). B, The working model is schematically represented. Ligand binding induces a conformational change to nuclear receptors (7 8 ), compatible with coactivator bindings but not with corepressor bindings (9 10 11 ). In principle, these two distinct conformations appear to be in dynamic equilibrium, which can be driven backward by corepressor binding and forward by ligand binding. Corepressor can either directly facilitate the conversion of a receptor from its holo to apo-state (I) or merely bind to a receptor after the dissociation of ligand from the holo-state (II). Our results appear to favor the former model.
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Based on these and other results (12, 13, 14), we propose that nuclear receptors are in a dynamic equilibrium between apo- and holo-states. In particular, corepressor binding appears to drive the equilibrium in the presence of ligand to the apo-state, whereas ligand binding, but not coactivator binding, is a major force to drive the equilibrium to the holo-state. It is important to note that our results can arise from two different models. First, corepressor can directly facilitate the conversion of a receptor from its holo to apo-state (Fig. 5B
, part I). Alternatively, corepressor can merely bind to a receptor after the dissociation of ligand from the holo-state (Fig. 5B
, part II). However, the fact that the CoRNR boxes alone introduced into the coactivator GRIP1 were not sufficient to affect the equilibrium in the presence of ligand (Fig. 4
) implies the importance of additional functional domains of SMRT. Thus, our results are consistent with the former model. One intriguing possibility that needs to be further investigated is the direct involvement of histone deacetylases, as suggested by the results with histone deacetylase inhibitor TSA (Fig. 3A
). Regardless of the detailed mechanism involved, however, our results strongly suggest that the increased ratio of corepressor to coactivator will effect the equilibrium to the apo-state in the presence of ligand. Thus, it is interesting to note that the expression level of corepressors and coactivators is modulated by various cellular signals (19, 20, 21), and that diverse modifications of coregulatory proteins, as exemplified with the CBP-dependent acetylation of ACTR (13), may also affect the equilibrium (21). Our results are consistent with the previously reported equilibrium model (22), in which the ratio of coactivator-corepressor bound to either receptor-agonist or antagonist complexes was proposed to regulate the dose-response curve. Our results also extend the findings of Schulman et al. (23), indicating that hormone modulates the equilibrium between active and repressive states of nuclear receptors. More recently, it was also shown that both ligand and corepressor binding have stabilizing effects on the structure of the receptors ligand binding domain (12). Therefore, we conclude that receptor conformation is far more dynamic than has been appreciated. More specifically, we suggest that the levels and activity of corepressors exert a direct effect on the extent of transcriptional activation in the presence of ligand. The dynamic interplay between corepressors and coactivators is likely to be widely applicable to other classes of transcription factors, as we and others (24, 25) have recently demonstrated for activating protein-1, nuclear factor-
B, serum response factor, and hepatocyte nuclear factor-1
.
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MATERIALS AND METHODS
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Plasmids
Mammalian expression vectors for Gal4/SRC-C, VP16/RAR, the full-length SMRT, SMRT-D, TRß, SRC-C, SRC-1, subGRIP1, ASC-2, and xSRC-3, bacterial expression vector for GST fusion protein to SRC-C, the reporter constructs Gal4-thymidine kinase (TK)-Luc, T3RE-TK-Luc and ß-RARE-TK-Luc, and the transfection indicator construct pRSV-ß-gal were as described (14, 16, 26, 27, 28, 29). PCR-amplified fragments encoding SRC-C and SMRT-D were inserted into EcoRI and XhoI restriction sites of His-tagging vector pET-28(a)+ and proteins were expressed and purified according to the manufacturers protocol (Novagen, Madison, WI).
Transfections
CV-1 cells were grown in 24-well plates with medium supplemented with 10% fetal calf serum for 24 h. Cells were transfected with 100 ng of LacZ expression vector pRSV-ß-gal and 100 ng of the indicated reporter gene, along with the indicated amounts of various mammalian expression vectors. Transfections and Luc assays were performed as described (16, 26, 27, 28, 29), and the results were normalized to the LacZ expression. The experiments were repeated at least three times, and the representative results were expressed as fold-activation (n-fold) over the values obtained with reporter alone, with the error bars as indicated.
GST-Pull Downs
The GST fusions or GST alone was expressed in Escherichia coli, bound to glutathione-Sepharose-4B beads (Amersham Pharmacia Biotech, Arlington Heights, IL), and incubated with labeled proteins expressed by in vitro translation by using the TNT-coupled transcription-translation system, with conditions as described by the manufacturer (Promega Corp., Madison, WI). Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and analyzed by SDS-PAGE and autoradiography.
Limited Proteolysis
TRß was in vitro translated/labeled with [35S]Met, and the digestions were performed for 10 min at room temperature with 10 µg/ml trypsin in the presence of the increasing amount of indicated proteins. The reaction products were analyzed by SDS-PAGE and autoradiography as described (15).
Ligand Bindings
GST fused to RAR was expressed in E. coli, bound to glutathione-Sepahrose 4B beads (Pharmacia, Piscataway, NJ) in binding buffer (25 mM HEPES, pH 7.6; 1 mM dithiothreitol; 300 µM phenylmethylsulfonyl fluoride; 1 mg/ml leupeptin; 1 mg/ml pepstain A; 20% glycerol; 20 mM NaCl; 0.2 mM EDTA; 1.5% BSA), incubated with 10 nM of [3H]9-cis-RA (0.13 µCi) in the presence or absence of indicated proteins at 4 C overnight, and extensively washed with washing buffer (binding buffer, 0.1% Triton X-100) and 1 ml of 50 mM Tris-Cl (pH 7.8). Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and counted for radioactivity.
Chromatin Immunoprecipitations and Western Analyses
293T cells were transfected with SMRT or SRC-1 expression vector and treated with 0.1 µM of 9-cis-RA for 12 h. Soluble chromatin from these cells was prepared and immunoprecipitated with a monoclonal antibody against SMRT (kind gift of Dr. Dean Edwards at University of Colorado Health Sciences Center), as recently described (14). The final DNA extractions were amplified 2125 cycles using pairs of primers that encompass the ß-RARE region and generate a 288-bp PCR fragment. The primers used were 5'-AAGCTCTGTGAGAATCCTG-3' and 5'-GGATCCTACCCCGACGGTG-3'. Western analyses were performed as previously described (26). The antibodies used were
HA monoclonal antibody (Roche Molecular Biochemicals, Indianapolis, IN, for SMRT fragments) and
GRIP1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
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ACKNOWLEDGMENTS
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The authors thank Drs. Christopher Glass and David D. Moore for helpful discussions, Dr. Mitch Lazar for subGRIP1 construct, and Dr. Dean Edwards for SMRT monoclonal antibody.
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FOOTNOTES
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1 Y.-C.S., S.-W.K., and S.L. made equal contributions to this study. 
This work was supported by grants from Postech Biotech Center (3PD02002) and GenoCheck, Inc.
Abbreviations: apo, Unliganded; ASC-2, activating signal cointegrator-2; CBP, cAMP response element binding protein-binding protein; GST, glutathione-S-transferase; HDACs, histone deacetylases; holo, liganded; Luc, luciferase; N-CoR, nuclear receptor corepressor; RAR, retinoic acid receptor; ß-RARE, retinoid-response element from the RARß2 promoter; SMRT, silencing mediator of retinoid and thyroid hormone receptors; SRC-1, steroid receptor coativator-1; TK, thymidine kinase; TR, thyroid hormone receptor; TSA, trichostatin A.
Received for publication April 19, 2002.
Accepted for publication December 9, 2002.
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