Characterization of Receptor Interaction and Transcriptional Repression by the Corepressor SMRT

Hui Li, Christopher Leo, Daniel J. Schroen and J. Don Chen

Department of Pharmacology and Molecular Toxicology University of Massachusetts Medical School Worcester, Massachusetts 01655-0126


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional regulation by steroid/thyroid hormones and retinoids is a critical component in controlling many aspects of animal development, reproduction, and metabolism (1, 2, 3, 4). The functions of these hormones are mediated by intracellular receptors, which comprise a large superfamily of ligand-dependent transcription factors (1). It has been established that both retinoic acid receptors (RARs) and thyroid hormone receptors (TRs) function via formation of heterodimeric complexes with retinoid X receptors (RXRs) (5, 6). Once bound to a DNA response element, the heterodimer responds to ligand through the C-terminal ligand-binding domain (LBD), which is known to mediate not only hormone binding but also receptor dimerization, transcriptional activation, and repression (7, 8).

Both TR and RAR can function as transcriptional repressors in the absence of ligands and potent activators upon binding of ligands (7). DNA-binding assays and functional analysis have demonstrated that the repressor activities of unliganded receptors depend on DNA response elements, as well as on the intact LBD of the receptors (7, 9, 10). In vivo, the TR/RXR heterodimer binds to DNA in the context of chromatin, and nucleosome assembly enhances the transcriptional silencing effect (11). Importantly, the oncogenic activity of v-erbA, a mutated form of TR, is directly linked to transcriptional repression (12, 13). In addition, deletion of the activation domain of RAR converts it into a potent transcriptional repressor, and this mutation was shown to cause defects in cellular differentiation and development (14, 15, 16). Therefore, transcriptional repression by unliganded nuclear receptors appears to play an important role in regulating cell growth and differentiation.

Hormone binding is thought to induce conformational changes that lead to ligand-dependent transformation of the receptors from repressors to activators (1). The C terminus of TR, about 20 amino acids, constitutes the 12th amphipathic helix (helix 12) of the LBD (17, 18, 19), which functions as a ligand-dependent activation core domain known as the AF2-AD, {tau}C, or {tau}4 domain (8, 20, 21, 22). Comparison of the LBD structures of the unliganded (19) and liganded receptors (17, 18) reveals a striking difference in the relative position of the helix 12/AF2-AD domain. This positional shift is thought to play an important role in receptor activation, allowing the liganded receptors to displace corepressors (8, 23, 24, 25) and to interact with coactivators (see reviews in Refs. 26–28).

SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors (24, 25) that are distinct from other proteins (29). They were shown to utilize the C-terminal domain for interaction with unliganded receptors (30, 31, 32, 33), and the N-terminal domain for transcriptional repression (25, 30). In this study, we investigate mechanisms of protein-protein interactions between SMRT and nuclear receptors and analyze the modes of repression mediated by SMRT/N-CoR. To do this, we define the interacting surfaces between SMRT and nuclear receptors in binding and functional assays. Next, we compare transcriptional repression mediated by SMRT and N-CoR using transient transfection assays in mammalian cells. Evidence is presented that SMRT and N-CoR interact with additional corepressors, and that histone deacetylation plays a role in SMRT/N-CoR- mediated repression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two Receptor Domains Are Essential for Interaction with SMRT
Deletion mutants in the carboxyl and amino termini of TR and RAR were used to analyze the contribution of different regions in the receptors for protein-protein interaction with SMRT. Figure 1AGo shows the domain structure of TR and the relative position of individual helices in the LBD as determined by x-ray crystallography (17, 18). The sequence at the C terminus region around helices 11 and 12 is also shown for both TR and RAR. [35S]Methionine-labeled TR or RAR deletion mutants were hybridized to glutathione S-transferase (GST)-SMRT and GST-RXR in far-Western analyses in the absence of hormone (Fig. 1BGo). The relative strengths of these interactions are summarized in Fig. 1CGo.



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Figure 1. Two Receptor Domains Interact with SMRT

A, Domain structure of human TRß and the sequences of the C-terminal helix 11 and 12 (AF2-AD) region of TR and RAR. The relative positions of individual helices determined by x-ray crystallography (18) are also indicated. B, Protein-protein interactions between receptors and SMRT or RXR in far-Western analyses. The full-length TR and RAR and their deletion derivatives were translated in vitro and labeled by [35S]methionine. All these deletion mutants expressed similar amounts of proteins as analyzed by SDS-PAGE and autoradiography (not shown). The position of GST-C.SMRT (SMRT) and GST-RXR (RXR) fusion proteins are as indicated (arrows). Please note that GST-RXR appeared as a doublet in our extract. C, Summary of relative levels of interactions between receptor mutants and SMRT or RXR. The relative levels of interactions were scored from background level (-) to strong (+++). nd, Not done. D, Human RARß and RAR{gamma} interact with SMRT in a ligand-reversible manner in far-Western blots. -, vehicle only; RA, 1 µM of all-trans-retinoic acid.

 
Full-length TR (1–456) associates well with both SMRT and RXR, and the interaction with SMRT can be drastically reduced upon hormone treatment. A residual weak interaction was observed in the presence of ligand, consistent with previous observations (24, 30). Carboxyl-terminal truncation at residue 441, which deletes helix 12, results in a mutant that interacts normally with RXR but that exhibits enhanced interaction with SMRT. Further truncation at residue 423, which removes part of helix 11, reduces the interaction with SMRT back to a level similar to that of wild type TR. In contrast, this deletion markedly reduces interaction with RXR. Further deletions that remove additional helices (helices 8, 9, and 10) result in barely detectable interaction with SMRT and no interaction with RXR. These results suggest that helix 12 inhibits SMRT association while helix 11 might promote the association.

Amino-terminal truncation of TR at residue 173, which removes the DNA-binding domain (DBD), does not affect the interaction with either SMRT or RXR. Further N-terminal deletion to residue 260, which removes the first and second helices of the TR LBD, markedly impairs SMRT association. No interaction with RXR by this mutant was detectable. Similarly, C-terminal deletion of helix 12 from RAR (1–403) also increases interaction with SMRT as compared with that of wild type RAR (1–462). Further deletion to residue 395, which removes part of helix 11, diminishes the enhanced interaction to a level comparable with that of full-length RAR, and ligand has little effect on the interaction. Together, these results identify two distinct interacting domains at the N-terminal hinge and C-terminal helix 11 regions of the receptor LBD that might act synergistically to promote interaction with SMRT. We find that the other two RAR isoforms, ß and {gamma}, also interact with SMRT in a ligand-reversible manner, although the interactions observed are weaker compared with that with RAR{alpha} (Fig. 1DGo). The interactions of both RARß and RAR{gamma} with RXR were not affected by ligand treatment.

Interaction of Helix 12/AF2-AD Deletion Mutants with SMRT in Yeast
To further understand the role of helix 12/AF2-AD in interaction with SMRT, we analyzed interactions between AF2-AD deletion mutants of RAR and RXR with C-terminal receptor-interacting domain of SMRT in a yeast two-hybrid system (Fig. 2Go). The RAR LBD alone is sufficient to interact with SMRT in a ligand-reversible manner (Fig. 2AGo, column 3), but the resulting activity is much weaker compared with that of full-length RAR (column 9). Similar to the far-Western results, SMRT and full-length RAR retain some interaction, even after treatment of the yeast cells with a saturating amount of ligand. It is unclear whether this obervation reflects an association between liganded receptors and SMRT or the existence of a small percent of unliganded receptors after ligand treatment. Deletion of the AF2-AD domain results in a RAR mutant that stimulates gene expression in response to hormone treatment in yeast (columns 4 and 10), as opposed to the dominant negative activity of this mutant observed in mammalian cells (14). The ligand-dependent activation of RAR403 is more obvious in the context of full-length receptor (column 10). A similar effect has been shown in v-erbA, which normally acts as a constitutive repressor in mammalian cells, but as a ligand-dependent activator in yeast (34). Cotransformation of the RAR403 mutants with a Gal4 activation domain-SMRT fusion (Gal4 AD-SMRT) strongly induces ß-galactosidase expression, even in the absence of hormone (columns 5 and 11). Furthermore, in contrast to the hormone-dependent dissociation seen with full-length RAR, hormone treatment does not interrupt these interactions. Similarly, the Gal4 DBD-SMRT fusion interacts strongly with the Gal4 AD-RAR403 mutants in a ligand-insensitive manner (columns 6 and 12). These results are consistent with the enhanced interaction observed in vitro and indicate that the AF2-AD domain may act as a negative regulatory element, controlling hormone-sensitive interaction between SMRT and nuclear receptors.



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Figure 2. Two-hybrid Interactions between SMRT and Helix 12/AF2-AD Deletion Mutants of Nuclear Receptors

A, Interaction between RAR403 and C- terminal domain of SMRT in yeast two-hybrid system. The indicated Gal4 AD and Gal4 DBD fusion constructs were cotransformed into yeast Y190 cells, and the resulting ß-galactosidase activities were determined from three independent colonies. The ß-galactosidase activities were determined in the absence (open bars) or presence (closed bars) of 1 µM of all-trans-RA. l, Ligand binding domain; f, full length; 403, RAR403 mutant with C-terminal truncation at residue 403. B, Interaction of SMRT with RXR443 and VDR in the absence of hormone (open bars) or presence (closed bars) of 1 µM 9-cis-RA (for RXR) or 100 nM 1,25 dihydroxyvitamin D3 (for VDR). 443, RXR443 mutant with C-terminal truncation at residue 443.

 
The effect of AF2-AD deletion in RXR on association with SMRT was also analyzed in the two-hybrid system (Fig. 2BGo). Ligand treatment weakly activates the Gal4 DBD-RXR LBD fusion (column 1), while cotransformation with Gal4 AD-SMRT enhances reporter gene expression (column 2), suggesting that SMRT can interact with RXR in either absence or presence of ligand. Truncation at residue 443 enhances the association between RXR and SMRT, and treatment with ligand does not alter this interaction (columns 4 and 5). These results suggest that SMRT can interact with RXR and that the AF2-AD domain of RXR also acts negatively in SMRT association. Furthermore, we observed a significant interaction between vitamin D3 receptor (VDR) and SMRT in the absence of hormone, and treatment with ligand reduces the interaction (column 8). This result is consistent with the recent finding that VDR also contains intrinsic transcriptional repression activity (35), suggesting that SMRT might mediate transcriptional repression by VDR.

Two SMRT Domains Mediate Differential Interactions with Nuclear Receptors
The finding that two regions of TR are essential for protein-protein interaction with SMRT suggests that SMRT might also contain duplicated receptor-interacting domains. Several deletion mutants of SMRT were used to test this possibility in a far-Western blot, and the results are summarized in Fig. 3AGo. The GST fusions of these SMRT mutants were overexpressed, and the purified proteins (Fig. 3BGo, lanes 1 and 2) or crude extracts (lanes 3, 4, and 5) were analyzed for interaction with 35S-labeled RAR and TR. SMRT(981–1495{Delta}) interacts equally well with both RAR and TR in the absence of ligands. RAR, but not TR, also interacts with degradation products of SMRT(981–1495{Delta}). Similarly, several fast migrating products of SMRT(1086–1291) also interact well with RAR, but not with TR (lane 4). These results indicate that RAR and TR may interact differently with SMRT. Consistent with this speculation, we find that SMRT(982–1291) (lane 2) as well as SMRT(1086–1291) interact more strongly with RAR than with TR. In contrast, the C-terminal fragment (1260–1495{Delta}) interacts better with TR than wth RAR (lanes 5). All these interactions were found to be sensitive to hormone treatment (Fig. 3BGo and data not shown). Together, these results identify two independent receptor interacting domains (RID-1 and RID-2) of SMRT that appear to display different affinities to TR and RAR.



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Figure 3. Two SMRT Domains Interact with the Receptors

A, Summary of SMRT deletion mutants used in this experiment and their relative levels of interaction with nuclear receptors in far-Western analyses shown in panel B. The amino acids encoded by the SMRT mutants are shown in parentheses. Bound-RAR and TR were detected by autoradiography, and the relative levels of interaction were scored from background level (-) to strong (+++). The column numbers in each panel correspond to constructs shown in panel A. Partially purified GST fusion proteins were used in lanes 1 and 2 and total cell extracts were used in lanes 3, 4, and 5. RID, Receptor interacting domain. + T3, Plus 1 µM T3; Q, glutamine-rich domain; H, putative helical region; {Delta}, an internal deletion between amino acids 1330 and 1375 resulting from alternative splicing.

 
Two SMRT Repression Domains
In addition to the C-terminal receptor interacting domains, SMRT/N-CoR proteins also contain strong transcriptional repression activity at their N-terminal regions. To define the minimal region needed for repression by SMRT, serial SMRT deletion mutants were generated, and their repression activities were analyzed using transient transfection (Fig. 4AGo). Consistent with previous observations, full-length as well as N-SMRT (amino acids 1–981) repress basal transcription strongly and in a dose-dependent fashion (rows 2 and 3), while C-SMRT (amino acids 982-1495{Delta}) exhibits minimal repression (row 4) compared with Gal4 DBD alone (row 1). Further deletion from the C terminus of N-SMRT reveals that amino acids 743 to 981 are not necessary for repression (row 5), while deletion to residue 475 reduces the repression effect about 2-fold (row 6). These results suggest that amino acids 475 to 981 may contribute in part to SMRT repression. Further C-terminal deletion to residue 337 drastically interferes with repression (row 7), indicating that the N-terminal boundary of this SMRT repression domain-1 (SRD-1) is located between amino acids 337 and 475. Truncation from the N terminus reveals that amino acids 1–134 are dispensable for repression by SRD-1 (row 8), while further deletion to residue 337 abolishes repression (row 9), indicating that the C-terminal boundary of the SRD-1 is within amino acids 134–337. When the SMRT fragment between amino acids 475 and 981 was tested for repression, we found that this fragment also strongly repressed basal transcription (row 10). Together with the observation that amino acids 743–981 are not important for repression, these results may define amino acids 475–743 as a second, independent SMRT repression domain (SRD-2).



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Figure 4. Multiple Transcriptional Repression Domains

A, Deletion mapping of the repression domains of SMRT. The transcriptional repression activities were analyzed by transient transfection in CV-1 cells. The relative levels of repression were determined from an average of three independent transfections using 0.1 µg (open bars), 0.2 µg (hatched bars), or 0.5 µg (closed bars) of plasmid DNAs. The starting and ending amino acids in each deletion construct are shown beneath each domain. SRDs, SMRT repression domains. B, Deletion mapping of the N-CoR repression domains (NRDs). The N-CoR domains are aligned with those of SMRT in panel A. The relative levels of repression were determined using 0.5 µg plasmid DNA and comparing the result to the Gal4 DBD alone. Two new transcriptional repression domains in N-CoR were found in addition to NRD-1 and NRD-2, which were identified previously (25). C, SDS-PAGE analysis of in vitro translated products of SMRT/N-CoR deletion constructs used in panels A and B. Two microliters of the in vitro translated products were analyzed in a 12.5% acrylamide gel, which was exposed overnight. Note that most of these constructs appear to produce doublet bands, perhaps due to secondary structure of the DNA used in the translation reaction. D, Western blot analysis of the repression-defective mutants of SMRT after transient transfection into 293 cells by using anti-Gal4 DBD monoclonal antibody (0.02 µg/ml) and detected by ECL kit. The gel on the left was resolved in a 12.5% acrylamide gel while the gel on the right was resolved in a 10% gel.

 
Sequence comparison between SMRT and N-CoR reveals that they share about 45% identity within both SRD-1 and SRD-2, suggesting potential functional conservation. Therefore, we tested whether the two SRD corresponding regions of N-CoR also contain repression activities. Consistent with a previous observation (25), amino acids 1–312 and 752-1016 of N-CoR exhibit strong repression activities (Fig. 4BGo, rows 2 and 3), and the two N-CoR domains corresponding to SRD-1 and SRD-2 also yield 10- to 30-fold repression (rows 4 and 5), similar to the repression effects observed by SRD-1 and SRD-2. These two additional N-CoR repression domains are termed N-CoR repression domain 3 and 4 (NRD-3 and NRD-4), and the two N-terminal repression domains are called NRD-1 and NRD-2. Together, these results indicate that both SMRT and N-CoR contain multiple, independent transcriptional repression domains.

To confirm that lack of repression in some of these SMRT/N-CoR deletion mutants is not due to lack of appropriate protein expression, we analyzed the expression of these constructs by both in vitro translation and Western blot analysis after transient transfection. We find that all constructs used in this experiment express approximately equal amounts of Gal4 DBD fusion proteins in vitro (Fig. 4CGo) and that the repression-defective mutants express well in vivo (Fig. 4DGo). These results indicate that lack of repression by certain SMRT/N-CoR deletion mutants are not due to lack of protein expression.

Multiple Mechanisms of Transcriptional Repression by SMRT/N-CoR
The mechanism of transcriptional activation by nuclear receptors has been shown to require recruitment of coactivators, including histone acetyltransferases such as CBP/p300 (36, 37, 38, 39). The opposite of histone acetylation, histone deacetylation, has recently been implicated in transcriptional repression by unliganded receptors and the associated corepressors. Several reports have described a corepressor complex containing a Mad-dependent corepressor mSin3A, a histone deacetylase HDAC1 or mRPD3, and the nuclear receptor corepressor SMRT/N-CoR (40, 41, 42, 43, 44, 45, 46, 47, 48). These results suggest that histone deacetylation may be a mechanism of transcriptional repression by unliganded receptors.

To confirm the interaction between mSin3A and the defined repression domains of SMRT and N-CoR, we tested the interactions between mSin3A and the individual repression domains of SMRT/N-CoR in a mammalian two-hybrid system. Coexpression of a VP16 AD-mSin3A fusion with all Gal4 DBD-SMRT/N-CoR repression domain fusions results in weak reduction of the repression activities (Fig. 5AGo). Coexpression of VP16 AD-mSin3A with a Gal4 DBD-HDAC1 fusion also results in partial release of repression mediated by Gal4 DBD-HDAC1 fusion. However, no activation above the background level was observed even though a VP16 activation domain was present. Since the weak interaction between SMRT/N-CoR repression domain with mSin3A in the two-hybrid system may reflect a dominant effect of repression over activation, we tested the interaction between mSin3A and individual SMRT/N-CoR repression domains in vitro by far-Western analysis. Full-length mSin3A was translated and labeled in vitro and used as a probe for GST fusions of various SRD and NRD domains. We find that mSin3A interacts specifically and consistently with NRD-1 and NRD-4 in this assay (Fig. 5BGo). In one experiment, we also detected interaction between SRD-2 and mSin3A (data not shown). No interaction is observed between SRD-1, NRD-2, and NRD-3. Therefore, these results suggest that different SMRT and N-CoR repression domains may repress transcription in a mSin3A-dependent or -independent manner.



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Figure 5. Multiple Mechanisms of Transcriptional Repression by SMRT and N-CoR

A, Two-hybrid interactions of mSin3A with SMRT and N-CoR repression domains and HDAC1. The indicated Gal4 DBD fusion of SMRT and N-CoR repression domains and HDAC1 were transiently transfected into CV-1 cells together with either Gal4 AD alone or Gal4 AD-mSin3A fusion as indicated. The relative levels of repression are expressed as the means of three independent experiments relative to the Gal4 DBD alone. B, In vitro protein-protein interactions between mSin3A and SMRT/N-CoR repression domains. The GST fusions of various SRD and NRD domains were expressed in Escherichia coli and partially purified. The GST fusion proteins were analyzed by SDS-PAGE (right bottom panel) and examined for their abilities to interact with 35S-labeled mSin3A in a far-Western blot (left upper panel). mSin3A appears to interact preferentially with intact GST-NRD-1 and GST-NRD-4 domains.

 
SMRT Represses Basal Transcription from Natural Promoters
The hypothesis that SMRT/N-CoR proteins are transcriptional corepressors that facilitate repression by unliganded receptors is supported by protein-protein interactions and transient transfections using the Gal4 fusion system. To provide further evidence that SMRT may be physiologically relevant in transcriptional regulation, we tested the effect of SMRT overexpression on transcriptional activity of receptor-responsive promoters. Overexpression of full-length SMRT (Fig. 6Go, lane 2), but not that of C-SMRT lacking the repression domains (lane 3), repressed basal expression from a mouse RARß2 promoter approximately 2-fold in comparison to the empty vector (lane 1). The same result is evident with two minimal response elements in the context of a thymidine kinase promoter in the absence of hormone (Fig. 5AGo). As expected, hormone treatment enhanced transcription from these promoters, while overexpression of full-length SMRT reduced slightly this ligand-dependent activation. C-SMRT enhances the ligand-dependent activation from these promoters (Fig. 5BGo). These results suggest that SMRT may, at least under certain circumstances, facilitate transcriptional repression of natural promoters.



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Figure 6. SMRT Represses Basal Transcription from RAR- and TR-Responsive Promoters

The mRARß2 promoter, two copies of the ßRARE (ßRARE-tk-luc), and the TRE (TRE-tk-luc) response elements were linked to a luciferase reporter and transiently transfected into CV-1 cells together with empty vector alone (lanes 1), full-length SMRT expression vector (lanes 2), or C-SMRT expression vector (lanes 3). The relative level of repression in the absence of hormone is shown in panel A, while the relative level of activation in the presence of 1 µM all-trans retinoic acid (atRA) is shown in panel B.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional repression has been recognized as a critical component of TR and RAR function and is thought to be mediated by association of unliganded receptors with silencing mediators (corepressors) such as SMRT and N-CoR. To understand the function of these putative corepressors, we have characterized their respective receptor interaction and transcriptional repression properties. Two distinct receptor-interacting domains of SMRT are identified that may interact directly with two corresponding regions in the receptor. We find that SMRT utilizes at least two distinct domains (SRD-1 and SRD-2) for transcriptional repression, consistent with a recent report (42). The two SRD-corresponding regions in N-CoR also repress basal transcription, indicating that N-CoR contains four independent repression domains. These results demonstrate the existence of multiple and possibly redundant receptor interaction and transcriptional repression domains in SMRT and N-CoR. One might expect that this multiplicity will ensure a reliable targeting of the corepressors and appropriate repression of target genes before activation.

The hinge region of TR was originally shown to interact directly with the RID-2 region of N-CoR (25). Our results indicate that TR requires an additional C-terminal region for efficient association with SMRT. Nested deletion analyses suggest that helix 11 of the TR LBD plays an important role in stabilizing SMRT association, presumably by cooperating with the N-terminal helix 1–2 region. The interaction of SMRT with either the N terminus or C terminus of the LBD alone is very weak but detectable, suggesting that these two potential interacting surfaces may act synergistically to promote protein-protein interactions and to ensure appropriate recruitment of the corepressors. Similarly, two independent regions in the receptor have been shown to act synergistically for interaction with N-CoR (32, 49, 50). It has recently been shown that a receptor dimer is required for interaction with SMRT/N-CoR and that SMRT/N-CoR may contribute to receptor-specific transcriptional repression (51). Furthermore, an antagonist of the transcriptional activation by RXR homodimer was shown to promote association with the corepressor SMRT (52). Together, these studies suggest that SMRT and N-CoR may utilize similar but distinct mechanisms for interaction with nuclear receptors.

We cannot exclude the possibility that the tight association with SMRT by the AF2-AD deletion mutants may weaken hormone binding to the receptor, but the ability of RAR403 to respond to ligand treatment in yeast cells indicates that this mutation does not eliminate the receptor’s hormone-binding capability, consistent with previous observations (14, 53). Therefore, the inability of hormone to dissociate corepressors is likely due to the lack of certain conformational changes that would normally take place in the presence of the AF2-AD. It is possible that the assumed shift of AF2-AD upon hormone binding is a prerequisite for additional structural changes that are important for corepressor dissociation. Alternatively, the shift of helix 12 may mask or compete with certain interacting surfaces required for binding corepressors. The fact that the AF2-AD deletion creates a mutant that binds tighter to the corepressors favors this model. We suspect that helix 11 could constitute such an interacting surface, since disruption of this helix eliminates the enhanced interaction resulting from deletion of AF2-AD. Our results suggest that AF2-AD may act to balance the association between nuclear receptors and the corepressors, by preventing overassociation of unliganded receptors with corepressors, thereby facilitating ligand-dependent dissociation of corepressors.

Nested deletion analysis reveals two distinct subdomains in SMRT that are capable of independent interaction with nuclear receptors. These two receptor- interacting domains, RID-1 and RID-2, interact differently with TR and RAR. The N-terminal RID-1 region interacts more strongly with RAR, and it contains a glutamine-rich domain, while the C-terminal RID-2 region interacts better with TR and contains a putative helical domain analogous to that identified previously in N-CoR (25). The different receptor-interacting properties of these two domains suggest that SMRT may utilize distinct mechanisms for interaction with different receptors. The RID-2 region in N-CoR has been shown to interact directly with the hinge region of TR (25), and therefore it is reasonable to predict that the N-terminal RID-1 region might interact with the C-terminal region of the LBD.

Functional analysis of the transcriptional repression activities of SMRT and N-CoR reveals two independent domains that are capable of repressing basal transcription. Together, there appear to be four independent repression domains in N-CoR and two in SMRT. These repression domains could act independently, and some repress basal transcription as efficiently as the full-length protein, suggesting that these domains might act redundantly and possibly through different mechanisms. Sequence comparison of these repression domains gives little clue as to possible mechanisms of repression. However, within SRD-1 and the corresponding NRD-3, four potential repeated motifs sharing a consensus sequence of GSITQGTPA have been identified (32). In addition, two other potential repeats with a consensus sequence of KGHVI•YEG are noted. These motifs are well conserved between SMRT and N-CoR, suggesting that they might contribute to repression.

Recently, several papers reported that mSin3A and the histone deacetylase HDAC1 form a ternary complex with SMRT and N-CoR (42, 46). These results indicate that SMRT and N-CoR, while interacting with unliganded receptors, can also interact with additional corepressors such as mSin3A and mSin3B (54), as well as the histone deacetylases HDAC1 (55) and mRPD3 (56). The recruitment of histone deacetylase to target promoters by unliganded receptors through SMRT, N-CoR, and mSin3 suggests that deacetylation of histones or other factors may play a role in transcriptional repression, perhaps by establishing an unfavorable chromatin structure for transcriptional activation (41). Our results suggest weak two-hybrid interactions between SMRT/N-CoR and mSin3A, or between mSin3A and HDAC1, even though a VP16 activation domain was present. Alternatively, these results may suggest that the repression activity of the corepressor complex is dominant over that of the VP16 activation domain. An in vitro protein-protein interaction assay detects association of mSin3A with NRD-1 and NRD-4, but not with other repression domains. Although our results are consistent with recent reports, our data also suggest the possibility of other repression mechanisms.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The GST fusions of C-SMRT (GST-SMRT) and RXR (GST-RXR) were described previously (24, 30). Serial C-terminal and N-terminal deletion mutants of human TRß and human RAR{alpha} were generated by appropriate restriction enzyme digestion and/or PCR amplification from the parental expression construct pCMX-hTRß and pCMX-hRAR{alpha} (57). The GST-SMRT deletion constructs were generated by enzyme digestion at indicated residues from the parental construct GST-SMRT. The Gal4 DBD fusions of individual repression domains of SMRT and N-CoR were generated by PCR amplification and were subsequently transferred to pGEX vector for expression of GST fusion proteins. The VP16 AD-mSin3A construct was created by subcloning the ScaI (at residue 56) to BglII fragment of mSin3A (58) into the pCMX-VP16 vector. Detailed information regarding these plasmids is available upon request.

Far-Western Analysis
GST fusion proteins were separated by denaturing protein gels (SDS-PAGE) and electroblotted onto nitrocellulose filters in transfer buffer (25 mM Tris-HCl, pH 8.3; 192 mM glycine; 0.01% SDS). After denaturation in 6 M guanidine hydrochloride (GnHCl), the proteins were renatured by stepwise dilution of GnHCl to 0.187 M in HB buffer (25 mM HEPES, pH 7.7; 25 mM NaCl; 5 mM MgCl2; 1 mM dithiothreitol). The filters were then saturated in blocking buffer (5% nonfat milk, then 1% milk in HB buffer plus 0.05% NP40) at 4 C overnight or at 37 C for 1 h. In vitro translated 35S-labeled proteins were diluted into hybridization buffer (20 mM HEPES, pH 7.7; 75 mM KCl; 0.1 mM EDTA; 2.5 mM MgCl2; 0.05% NP40; 1% milk; 1 mM dithiothreitol), and the filters were allowed to hybridize overnight at 4 C. After three washes (5 min each) with the hybridization buffer, the bound proteins were detected by autoradiography.

Yeast Two-Hybrid Assay
The yeast two-hybrid assay was carried out in the Y190 yeast strain (59). The Gal4 DBD fusion constructs were generated in either the pAS or pGBT vector (CLONTECH, Palo Alto, CA), and the Gal4 AD fusion constructs were in the pGAD or pACT vector (CLONTECH). The ß-galactosidase activities were determined with the O-nitrophenyl ß-D-galactopyranoside (Sigma, St. Louis, MO) liquid assay as previously described (30).

Cell Culture and Transient Transfection
African green monkey kidney CV-1 cells were grown in DMEM supplemented with 10% resin-charcoal stripped FBS, 50 U/ml penicillin G, and 50 µg/ml streptomycin sulfate at 37 C in 5% CO2. One day before transfection, cells were plated in a 24-well culture dish at a density of 50,000 cells per well. Transfection was performed by standard calcium phosphate precipitation (57). All transfection experiments were performed in triplicate and were replicated at least once. Twelve hours after transfection, cells were washed with PBS and refed fresh medium containing indicated amounts of ligands. After 30 h, cells were harvested for ß-galactosidase and luciferase assay as described previously (30). The relative luciferase activities are arbitrary light units normalized to the ß-galactosidase activities.

In Vitro Translation and Western Blot
In vitro transcription/translation reactions were carried out in rabbit reticulocyte lysates using the TNT T7 Quick coupled transcription/translation system (Promega, Madison, WI). [35S]Methionine (Amersham, Arlington Heights, IL) was added during the translation reactions, which were performed at 30 C for 90 min. The translated reactions were analyzed by SDS-PAGE, followed by autoradiography. For Western blot analysis, transfected cells were lysed in SDS-sample buffer, and the extracts were separated by SDS-PAGE. The gels were transferred onto nitrocellulose membranes, blocked with nonfat milk, and hybridized with anti-Gal4 DBD monoclonal antibody according to manufacturer’s recommendation (Santa Cruz Biotechnology, Santa Cruz, CA). The filters were washed and incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody and developed by enhanced chemiluminescent reaction (Amersham).


    ACKNOWLEDGMENTS
 
The authors thank Drs. Neal C. Brown, Kristin Carlson, Carol Mulder, Douglas R. Waud, and George Wright for critical reading and insightful suggestions of this manuscript. We are grateful to Drs. Horlein and M. G. Rosenfeld for providing the N-CoR plasmid. We are especially grateful to Christian A. Hassig and Dr. Stuart L. Schreiber for providing the Gal4 DBD-HDAC1 plasmid, and Drs. Ronald A. Depinho and Margaret S. Halleck for providing the mSin3A construct. Part of the data presented was initiated by J.D.C. in Dr. Evans’ laboratory.


    FOOTNOTES
 
Address requests for reprints to: J. Don Chen, Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655-0126.

This work was supported by an American Society of Hematology Junior Faculty Scholar Award and the USAMRMC Breast Cancer Research Program Idea Award BC961877 (to J.D.C.) and an Arthritis Foundation postdoctoral fellowship (to D.J.S.).

Received for publication May 20, 1997. Revision received August 29, 1997. Accepted for publication September 5, 1997.


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