Determination of Nuclear Receptor Corepressor Interactions with the Thyroid Hormone Receptor
Anita Makowski,
Sabrina Brzostek,
Ronald N. Cohen and
Anthony N. Hollenberg
Thyroid Unit (A.M., S.B., A.N.H.), Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215; and Section of Endocrinology (R.N.C.), Department of Medicine, University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Anthony N. Hollenberg, M.D., Division of Endocrinology, Beth Israel Deaconess Medical Center, Research North, 330 Brookline Avenue, Boston, Massachusetts 02215.
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ABSTRACT
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The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.
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INTRODUCTION
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THE THYROID HORMONE receptor (TR) isoforms, members of the nuclear hormone receptor superfamily, regulate target gene expression in response to their ligand T3 (1, 2). In the absence of ligand, the TR
1 and TRß1 isoforms are potent transcriptional repressors because of their ability to recruit the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT) (3, 4, 5). When bound to the TR the corepressors recruit a multiprotein complex including the histone deacetylases and other proteins such as the WD-40 protein TBL-1, which lead to active repression on thyroid hormone response elements (TREs) (6, 7, 8, 9, 10, 11). The addition of T3 causes a conformational change in the ligand-binding domain of the TR and leads to the release of the corepressor complex and the recruitment of members of the coactivator family, which leads to transcriptional activation through histone acetylation and methylation (12).
NCoR and SMRT are modular proteins that contain N-terminal repression domains and C-terminal nuclear receptor interaction domains (IDs) (13, 14, 15). Originally, both NCoR and SMRT were described to contain two nuclear receptor IDs (N2 and N1, S2 and S1, respectively; see Fig. 1
) but more recently it has become clear that NCoR possesses a third domain, N3. N3 is N terminal from the two conserved domains and is not present in SMRT. Furthermore, N3 interacts only with the TR and appears not to bind other nuclear receptors (16, 17). Each of the nuclear receptor IDs in both NCoR and SMRT are characterized by the presence of motifs termed CoRNR boxes (I/LxxI/VI), which are essential for binding to nuclear hormone receptors (NRs) (18). In addition, an extended helical domain (LxxH/IIxxxI/L) has been proposed to characterize each of the IDs present in NCoR and SMRT (19, 20). Surprisingly, by inspection, this helical motif is lacking in N3 suggesting that corepressor interactions with the TR may be different. While the IDs present in NCoR and SMRT show much homology, they mediate interactions with specific nuclear receptors. Indeed, the TR prefers to recruit NCoR, and this specifically requires the presence of N3 while the orphan receptor Reverb also recruits NCoR only but requires amino acids immediately N terminal to the CoRNR box of N2 to do so (17, 21). In contrast, SMRT prefers to recruit the retinoic acid receptor (RAR) isoforms and that specificity is mediated by the CoRNR box sequence of S2 (17, 21). Furthermore, SMRT also can recruit peroxisomal proliferator-activated receptor (PPAR)
and PPAR
when bound to an antagonist but through the S1 domain only. In addition, S1 and N1 also bind retinoic X receptor (RXR) well and mediate interactions with heterodimeric bound complexes such that N1 or S1 bind to the RXR component while the N-terminal IDs interact with the partner (22, 23, 24).

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Figure 1. Schematic Structure of NCoR, its Isoforms, and Deletion Mutants
Full-length NCoR contains three repressing domains (RD1, RD2, and RD3) and three IDs (N3, N2, and N1) whose amino acid boundaries are shown. NCoRI is a truncated NCoR isoform that lacks the RDs but contains the IDs. It is translated both in vitro and in vivo from an endogenous ATG. NCoRITM contains a mutation from an isoleucine to an alanine in the first isoleucine of each of the CoRNR boxes present in the NCoR IDs. NcoRII-V contains a valine mutation in N3 and N2 that is N terminal to the CoRNR box. All mutants are otherwise identical in structure to NCoRI.
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Corepressor specificity is further complicated by the nature of the NR dimer bound to a specific response element. For example, the TR homodimer binds both NCoR and SMRT more avidly than the TR-RXR heterodimer while the RXR/RAR heterodimer appears to be necessary for the recruitment of corepressor (25, 26, 27). We have hypothesized that NCoR is still strongly preferred by the TR homodimer because of the cooperative binding of a single NCoR molecule to the TR homodimer via N3 and N2 while the RAR-RXR heterodimer recruits SMRT through S2 and S1, respectively (17, 26).
The recruitment of either NCoR or SMRT is mediated by the ligand-binding domain of NRs. Mutagenesis studies performed on the TR suggest that the corepressor binding surface overlaps the coactivator binding surface with the primary contact site being comprised of residues in helix 3 and helix 5 (21, 28). In the presence of ligand this region is normally blocked by helix 12, but in the absence of ligand helix 12 is shifted to allow for corepressor binding. Other sites that influence corepressor binding include a region adjacent to helix 1 and a portion of helix 11. Recently, the crystal structure of antagonist-bound PPAR
coupled to the S1 domain of SMRT has been reported. S1 contains the extended helical motif and indeed forms a three-turn
-helix, which makes contact with residues in helices 3, 4, and 5 (23). Interestingly, many of these residues are not conserved in the ligand-binding domain (LBD) of other NRs and suggest that if the structure of the LBD and corepressor ID are sufficiently altered, the recruitment of corepressor may be substantially altered as well.
In the following study we have further analyzed the preference of NCoR for the TR homodimer and demonstrate that NCoR prevents dissociation of the TR from thyroid hormone response elements. In addition, we have further investigated the binding specificity of N3, which lacks an extended helical motif, and N2 and determined that specific amino acid residues are necessary within the IDs for the recruitment of the TR.
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RESULTS
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NCoR Stabilization of the TR in Solution Requires N3 and N2
We and others have recently demonstrated that NCoR possesses a third ID termed N3 that lies N terminal to the two relatively conserved domains (N2 and N1), which are also present in SMRT (16, 17). The location of these domains is depicted in Fig. 1
. To test the importance of this domain as well as N2 and N1 in mammalian cells, we asked whether it affected the ability of NCoR to stabilize the TR homodimer in solution, as we have shown previously (14). To do this, we used a mammalian two-hybrid system in which the TR-LBD is fused both to the Gal4-DNA binding domain and the VP16 activation domain in two separate plasmids. As shown in Fig. 2
, overexpression of NCoRI, a truncated isoform of NCoR which lacks the repression domains, allows for the TR-LBDs to interact in solution. In contrast, in the absence of overexpressed NCoR, the TR homodimer does not form. In addition, overexpression of an analogous truncated isoform of SMRT does not allow the homodimer to form (Ref. 14 and data not shown). Mutation of the first isoleucine to an alanine in each of the three CoRNR boxes (IxxII to AxxII) was then introduced into NCoRI, either alone or in combination, and the ability of these mutants to stabilize the homodimer was then tested. Mutation of N3 completely blocked stabilization as did mutation of any two or all three (TM) of the CoRNR boxes. Mutation of N2 alone also preserved some stabilizing activity but only at a marginal level. As expected, mutation of N1 alone had a weaker effect on stabilization as did mutation of either N2 or N3. The N1 mutant, however, was not as effective in promoting stabilization as the wild-type protein, suggesting that N1 could play a role in this process. However, we have previously shown, using EMSA and transfection studies, that N1 is not required for the recruitment of NCoR by the TR to a TRE, suggesting that the effect seen here may be unique to the assay used (17). In summary, these data demonstrate that the TR homodimer is stabilized by NCoR and preferentially interacts with a single NCoR molecule through two IDs, likely N3 and N2. Furthermore, these data suggest that one of the main functions of NCoR might be to stabilize TR homodimer binding to its target response element.

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Figure 2. NCoR Stabilizes the TR Homodimer in Mammalian Cells via N3 and N2
CV-1 cells were transfected as described with Gal4-TRß1 and VP16-TRß1 expression vectors and the UAS-TK-Luc reporter. In addition, cells were cotransfected with wild-type pKCR2-NCoRI (WT) or identical amounts of the indicated NCoRI CoRNR box mutant construct (i.e. N3 indicates that the N3 CorNR box has been mutated as shown). The data are expressed as fold interaction where 1 represents the interaction of Gal4-TR and VP16-TR in the absence of cotransfected NCoRI. This value was equal to that obtained with Gal4-TR alone.
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NCoR Stabilizes the TR Homodimer Complex on a Target Hormone Response Element
To test the hypothesis that NCoR may stabilize the TR homodimer on a thyroid hormone response element, we employed EMSA. In initial experiments we examined the ability of TRß homodimers to associate with a DR+4 response element in the presence of a nonspecific competing oligonucleotide [in this case a signal transducer and activator of transcription (STAT) response element]. As is shown in Fig. 3B
, the TR homodimer binds well after a 20-min incubation to a radiolabeled DR+4 probe (lane 2). The addition of subsequent samples was performed at the indicated time point to a running gel, which accounts for the different mobility of similar complexes. Addition of excess nonspecific oligonucleotide competitor does not dissociate the complex, and specific binding remains stable after the addition of cold competitor (lanes 35). When NCoRI is added, a supershifted complex is seen (lane 6) and, similar to the TR homodimer, it remains stable after the addition of excess cold competitor. Thus, the addition of NCoR does not affect the association of the TR complex to its response element. Similar results were seen in the absence of a nonspecific competitor.

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Figure 3. NCoR Stabilizes the TR Homodimer on a DR+4 Response Element
EMSAs were carried out to assess the role of NCoR in stabilizing TR binding to a DR+4 response element as is shown schematically in panel A such that specific TR homodimer binding is competed with either excess cold DR+4 or a nonspecific binding site. B, Identical amounts of in vitro translated (IVT) TRß1 (lane 2) and TRß1 with NCoRI (lane 6) were incubated with radiolabeled DR+4 for 20 min at room temperature. Subsequently, 50-fold excess of a nonradiolabeled STAT binding site (cold probe) was added to lanes 35 and lanes 79, and loading was carried out at the indicated time points. The arrows indicate the homodimer or TR/NCoR complex. Lane 1 contains unprogrammed reticulocyte lysate. C and D, An identical paradigm to that used in panel B was employed except that the cold probe used was 50-fold more than the specific DR+4 competitor. Lanes 2 and 7 contain TRß1 or TRß1 with NCoRI in the absence of cold probe, respectively. Lane 1 contains unprogrammed reticulocyte lysate. After the standard 20-min incubation with the radiolabeled DR+4 probe, excess cold probe was added, and loading was carried out at the indicated time points. Densitometry was performed on all EMSAs where maximal binding (100%) was set in the absence of cold competitior (lanes 2 and 7). E, Supershift experiments were performed using specific anti-TRß and NCoR immunoserum. In the right panel increasing amounts (either 1, 2, or 3 µl) of an affinity-purified NCoR antibody were added. The smaller arrows indicate the supershifted complexes.
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We next examined the dissociation of the TR homodimer by adding excess nonlabeled DR+4 after the standard 20-min incubation. In these experiments the TR (and, where indicated, NCoR) was incubated with labeled DR+4 for 20 min. After this incubation period, 50-fold excess nonlabeled (cold) DR+4 was added to each tube for the indicated period of time. Subsequent samples were loaded at the indicated time points to a running gel. After the film had been exposed, all bands were quantitated using densitometry. In the presence of excess cold DR+4, we found that the TR homodimer was quickly dissociated from the labeled DR+4 (as early as 2 min; compare lanes 2 and 3 of Fig. 3C
) and that this dissociation reached a maximal level by 20 min (lane 6, Fig. 3C
; and lane 4, Fig. 3D
). In contrast, the TR/NCoR complex was not competed for to an appreciable degree by excess DR+4 when examined from 2 to 60 min after the initial 20-min incubation time (Fig. 3
, C and D). Similar results were seen with a range of excess cold probe (data not shown). These results are consistent with NCoR stabilizing the TR homodimer and preventing the complex from actively dissociating from its target response elements. Thus it is likely that NCoR, through the N3 and N2 IDs, is able to stabilize the TR homodimer to DNA, which may play an important role in the ability of the unliganded TR to mediate transcriptional repression.
Interestingly, the addition of NCoR to the TR appears to reduce the overall bound complex to the DR+4 probe (compare lanes 2 and 7 of Fig. 3
, panels B and C). To confirm the presence of both the TR and NCoR in the complex, we used both TR- and NCoR-specific antiserum in supershift assays. As shown in Fig. 3E
(left panel), the TR antibody fully supershifts both TR and NCoR-TR complexes, while the NCoR antibody (right panel, Fig. 3E
) blocks formation of the TR-NCoR complex only and weakly supershifts it. The NCoR antibody does not affect the remaining TR homodimer. Thus, the TR and NCoR are, as expected, present in this complex. These data imply that the perceived decrease in bound complex is due to the addition of cofactors such as NCoR, which alters the visualized complex but not its components.
Given the ability of NCoR to stabilize TR binding to its response element, we next compared its action to that of RXR, which has long been known to enhance TR binding through formation of a RXR-TR heterodimer. Indeed, the RXR-TR complex associates in a similar fashion to the TR homodimer and TR-NCoR complex in that its binding affinity remains stable over time (compare lane 2 with lanes 7 and 8 in Fig. 4B
). However, unlike the TR homodimer but similar to the TR-NCoR complex, dissociation of the RXR-TR heterodimer does not occur after the addition of excess cold probe at the indicated time points after the initial 20-min incubation (lanes 36, Fig. 4B
). This result is consistent over a wide range of excess cold probe for RXR (Fig. 4C
, lanes 711). In contrast, the homodimer is competed for by very low amounts of cold probe both in isolation (Fig. 4C
, lanes 36) and in the presence of limiting amounts of RXR (lanes 711, lower band). Thus, NCoR appears to have a similar function to RXR and allows for enhanced stability of the TR on DNA.

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Figure 4. RXR Stabilizes the TR-Complex on a DR+4 Response Element
A, RXR prevents dissociation of the TR on a DR+4 element. B, EMSA was carried out with in vitro translated (IVT) TRß1 (lane 1) or IVT TRß1 and RXR (lane 2) and a radiolabeled DR+4 probe for 20 min. After the standard incubation 50-fold excess cold DR+4 was added, and loading was carried out at the indicated time points (lanes 36). In addition, cold nonspecific competitor was also added, and loading was carried out at the indicated time points onto a running gel (lanes 7 and 8). Densitometry was carried out with 100% binding set on lane 2. C, Increasing amounts of cold DR+4 (CP) were added to either preincubated TR or TR/RXR reactions with a radiolabeled DR+4 probe. The cold competitor was allowed to compete for 20 min after which the samples were loaded. Densitometry was performed with lane 2 serving as 100% for the TR homodimer, and lane 7 serving as 100% for the TR-RXR heterodimer. Lane 1 contains unprogrammed reticulocyte lysate. The fold amount of excess cold probe used was either 5-, 10-, 50-, or 100-fold as depicted.
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The Specificity of NCoR for the TR Is Mediated by an Extended Helical Motif
The interaction of corepressors with the TR is felt to be mediated by a helical motif centered around the CoRNR box present in each of the IDs. This can be demonstrated by the introduction of helix-breaking amino acids as shown in Fig. 5A
where mutation of an aspartate to a proline blocks binding of N2 to the TR homodimer. In contrast, introduction of a more conservative alanine residue, which would not disrupt the helix, only marginally decreases the binding of N2 to the TR homodimer. Inspection of N3 reveals that it also possesses amino acids, both an asparagine and phenylalanine, in the region surrounding the CoRNR box, that are conducive to helix formation. To confirm this we mutated N3 in the context of all of NCoRI because, unlike N2, mutations of the N3 IxxII motif alone severely disrupt binding of NCoRI to the TR. As shown in Fig. 5B
, NCoRI binds well to the TR homodimer and supershifts the homodimer. Mutation of the first isoleucine in the CoRNR box (N3M, lanes 4 and 5) disrupts most binding to the TR as we have shown previously. Mutation of the phenylalanine, which would be predicted to be important for formation of a helix, to an alanine significantly impairs TR binding (N3F-A, lanes 6 and 7) but still allows for some binding. In contrast, changing the phenylalanine to a proline (N3F-P, lanes 8 and 9), which would disrupt formation of a helix, blocks most binding. All of the NCoRI proteins were synthesized in equal amounts (data not shown). Thus, despite its apparent lack of homology to the other IDs outside of the region of the CoRNR box, N3 also forms a helical domain that appears to be TR specific.

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Figure 5. A Helical Structure Is Present in the Corepressor IDs
A, Alignment of the CoRNR box regions surrounding N3, N2, and S2 reveals a conserved CoRNR box motif that likely represents a portion of a helical motif. To test the importance of this helix, a D-to-P mutation (helix breaking) or D-to-A mutation (helix stabilizing) were introduced into the N2 domain, and resulting GST proteins were incubated with in vitro translation (IVT) TRß1 and a radiolabeled DR+4 probe. Lane 1 contains TRß1 alone; lanes 2, 4, and 6 contain 20 ng of GST fusion protein; lanes 3, 5, and 7 contain 50 ng of GST protein. The arrows indicate the homodimer or TR-NCoR complex. B, Mutations were introduced into the indicated amino acid in N3 in the context of NCoRI, and proteins for EMSA were produced by IVT and incubated with IVT TRß1 and a radiolabeled DR+4 probe. Lanes 2, 4, 6, and 8 contain 1.5 µl of IVT NCoRI or its mutants while lanes 3, 5, 7, and 9 contain 3 µl. The arrows indicate the homodimer or TR-NCoR complex.
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Although both N2 and S2 in SMRT appear to have the extended helical motif (LxxH/IIxxxI/L) described by Perissi et al. (19) (see Fig. 6A
), N3 does not in that the leucine present at position 3 is an alanine in N3. However, alignment of N3 and N2 demonstrates the presence of a conserved isoleucine immediately adjacent to a tyrosine present in both (position 1 in Fig. 6A
). Interestingly, this isoleucine is a valine residue in S2, which binds the TR quite weakly (Fig. 6A
), and neither N1 nor S1, which bind very weakly if at all to the TR, possesses an isoleucine at this location. To test the role of this residue in mediating both N3 binding and specificity for the TR, we mutated it both to an alanine and a valine. As shown in Fig. 6B
, the wild-type N3 ID interacts strongly with the TR homodimer. Increasing amounts of protein diminish the amount of TR homodimer present (lanes 26). In contrast, N3 I-A (at position 1) does not bind to the TR homodimer (lanes 710), which suggests that this isoleucine is essential for the function of N3 and potentially allows N3 to form a helix. We next tested the N3 I-V mutation and found that binding of this mutant was severely impaired both in the context of the NCoR-TR supershift and removal of the homodimer (lanes 1114), indicating the importance of this residue in allowing for specific TR binding. This is supported by a similar mutation in N2 that also impairs its ability to bind well to the TR homodimer (Fig. 6C
, compare lanes 36 with lanes 710). To confirm that the isoleucine at position 1 is important in mediating specificity for the TR, we changed the valine present in S2 at this position to an isoleucine. Indeed, recruitment of S2 by the TR homodimer was increased both in the context of the supershift and diminution in homodimer binding (Fig. 6D
). However, S2, even with the added isoleucine, is a weaker binder than either N3 or N2 as the protein concentrations required for binding are 20-fold higher. Taken together, these results demonstrate that the specificity of NCoR for the TR is mediated, in part, by an extension of the proposed helical domain maintained by each of the corepressor IDs that interact strongly with the TR.

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Figure 6. An Extended Isoleucine-Rich Motif Allows the TR to Recruit Individual IDs
A, Alignment of N3, N2, and S2 revealed the presence of a conserved isoleucine in N3 and N2 just beyond the helical domain (HM) first demonstrated by Perrissi et al. (19 ). EMSAs were performed to study its importance in the recruitment of NCoR by the TR. B, In vitro translated (IVT) TRß1 was incubated with a radiolabeled DR+4 probe and increasing amounts of GST-N3 or GST N3 containing an I-A (lanes 710) or an I-V (lanes 1114) mutation in the indicated region of N3. Lanes 3, 7 and 11: 5 ng of GST protein; lanes 4, 8, and 12: 10 ng of GST protein; lanes 5, 9, and 13: 20 ng of GST protein; lanes 6, 10, and 14: 50 ng of GST protein. C, IVT TRß1 was incubated with a radiolabeled DR+4 probe and increasing amounts of GST-N2 or GST N2 containing an I-V mutation in the indicated region of N2. The amounts of protein used were identical to those in panel B. D, IVT TRß1 was incubated with a radiolabeled DR+4 probe and increasing amounts of GST-S2 or GST S2 containing a V-I mutation in the indicated region of S2. Lane 1: unprogrammed reticulocyte lysate; lanes 2 and 5: 50 ng of GST protein; lanes 3 and 6: 100 ng of GST protein; lanes 4 and 7: 200 ng of GST protein.
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Whereas the above data demonstrate the importance of position 1 in the context of individual IDs, we wanted to ensure that it was equally important in the context of all of the NCoR IDs. To do this we employed in vitro translated NCoRI, which contains all three IDs in EMSA. Mutation of each of the CoRNR boxes in NCoRI (TM) or those present in N3 and N2 causes loss of all NCoR binding to the TR (lane 3 in Fig. 7A
and lanes 36 in Fig. 7B
). Mutation of the isoleucine at position 1 to either an alanine (Fig. 7A
) or valine (Fig. 7B
) in N3 and/or N3 and N2 together dramatically reduces NCoR binding. All binding is not lost in the context of the alanine mutation present in N3 alone, in that the original helical motif is still present in N2 and N1, which allows for some binding but the TR homodimer is still well preserved indicating that the binding is weak. Similarly, the valine mutations still show some binding consistent with a preserved N1 and partially preserved N3 and N2 domains. However, the initial conserved isoleucine in N3 and N2 appears to be critical for TR specificity in the context of all of the NCoR IDs. Each of the NCoR constructs used was translated with equal efficiency in vitro as seen for the valine mutations shown in Fig. 7C
.

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Figure 7. NCoR Requires the Extended Isoleucine-Rich Motif to Interact with the TR
A, EMSA was performed with in vitro translated (IVT) TRß1 and IVT wild-type NCoRI (lanes 5 and 6) or NcoRITM (lane 4) or NCoRI I-A mutants within N3 (lanes 7 and 8) or N3 and N2 (lanes 9 and 10). Either 3 µl (lanes 4, 5, 7, and 9) or 5 µl of IVT NCoRI (lanes 6, 8, and 10) were incubated with identical amounts of IVT TRß1. B, A similar paradigm was employed with NCoRI containing the I-V mutations in N3, N2, or both. Either 3 or 5 µl of IVT NcoRI were used. Lanes 3 and 4 contain the double CoRNR box mutant while lanes 5 and 6 contain the triple mutant shown in Fig. 1 . C, WT NCoRI and its mutants were IVT in the presence of 35S-methionine, and the resulting products were resolved on SDS-PAGE.
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We next examined the ability of the NCoRI mutants shown above to interact with the TR in mammalian cells using a two-hybrid assay. As we have shown previously, mutation of the N3 CoRNR box by introducing an alanine at position 8 causes more than an 80% reduction in TR recruitment in mammalian cells. In contrast, mutation of the N2 CoRNR box with an alanine at position 8 alone has only a 5060% effect. Mutation of the proximal isoleucine at position 1 to a valine in N2 has a similar effect as mutating the N2 CoRNR box whereas a similar mutation in N3 has a more severe effect but does not approach the severity of the N3 CoRNR box mutation. This is consistent with the now present valine allowing for marginal retention of the tertiary structure of N3. A double-mutation in position 1 in both N3 and N2 comes closest to approaching the N3 CoRNR box mutation. In contrast, substitution of the proximal isoleucine with a valine does not affect recruitment of RAR
and appears to slightly enhance RAR
recruitment as is shown in Fig. 8A
. This demonstrates that the mutant NCoR constructs are expressed well and supports earlier work by us and others that showed that recruitment of RAR
was dependent significantly upon the sequence of the CoRNR box itself and not surrounding sequences. Furthermore, N3 plays no role in the recruitment of RAR
as it is dependent only on N2 and indeed prefers the S2 present in SMRT. To confirm that each of the Gal4-NCoR chimeras was expressed at similar levels in CV-1 cells, we immunoblotted nuclear extracts prepared from transfected cells with an NCoR antibody directed against a C-terminal peptide. As shown in Fig. 8B
, relative levels of expression are similar except for the N3 and N2 CoRNR box mutants, which were slightly overexpressed. Thus, decreased levels of expression of the Gal4-NCoRI chimeras does not explain the defective actions of the mutant NCoRs on the recruitment of the TR.

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Figure 8. The Extended Isoleucine-Rich Motif Is Essential for Interactions with the TR in Mammalian Cells
CV-1 cells were transfected with expression vectors for Gal4-NCoRI or the indicated mutants in the context of NCoRI and VP16-TRß1 or VP16-RAR and the UAS-TK-Luc reporter. The data are expressed as percent maximal interaction where 100% represents the interaction between Gal4-NCoRI (WT) and VP16-TRß1 or VP16-RAR . B, Nuclear extracts were prepared from CV-1 cells transfected with either Gal4 alone or the indicated Gal4-NCoRI constructs. Equal amounts of nuclear extract were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with a specific NCoR antisera. The visualized band represents a species with a molecular mass of approximately 165 kDa.
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Finally, if the conserved proximal isoleucine present in N3 and N2 is important for TR specificity, we would expect that NCoR action on a TRE would be impaired in the presence of either the valine or alanine substitutions. To confirm this we assessed the ability of an inhibitor of endogenous NCoR, termed NCoRI, to block repression by TRß1 on a DR+4 element. In the absence of ligand, TRß1 represses the activity of the DR+4 reporter by 2.5-fold (Fig. 9
). Cotransfection of NCoRI, which contains N3, N2, and N1 but lacks any of the repression domains, completely reverses repression. Cotransfection of a NCoRI molecule that contains mutations in each of the ID CoRNR boxes is unable to reverse repression, despite being expressed at equivalent levels in CV-1 cells (Fig. 9
). Similarly, cotransfection of an NCoRI molecule with disrupted N3 and N2 CoRNR boxes fails to reverse repression to any appreciable degree. Substitution of the conserved proximal isoleucine with a valine or alanine in both N3 and N2 in the context of NCoRI, were also defective in their ability to reverse repression induced by endogenous NCoR, despite being expressed at equivalent levels to NCoRI in CV-1 cell nuclear extracts. Their effect was similar to that seen in EMSA in which a complete loss of function was not achieved. These data are consistent with the importance of this residue in the recruitment NCoR by the TR on a TRE and suggest that the helical surface of N3 is specific for the TR LBD.

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Figure 9. The Extended Isoleucine Motif Is Essential for Normal NCoR Function on a TRE
CV-1 cells were transfected with a DR+4-Luc reporter, TRß1, and either wild-type NCoRI or the following mutant NCoRIs: 1) The triple CoRNR box mutant (TM); 2) the double CoRNR box mutant (N3, 2); 3) the I-V mutation in both N3 and N2; and 4) the I-A mutation in both N3 and N2. The data are displayed as fold repression (fold repression is the level of repression achieved in the presence of TRß1 only as compared with an empty expression vector). To ensure that NCoRI and its mutants were expressed at similar levels, a Western blot was performed on nuclear extract from transfected cells with a specific NCoR antisera. The visualized band is approximately 130 kDa. No band was visualized in mock-transfected cells.
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DISCUSSION
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The recruitment of the corepressors NCoR and SMRT is critical for the action of nuclear receptors in the absence of ligand and in the presence of antagonists, as it allows for the subsequent recruitment of a multiprotein complex that mediates transcriptional repression. The physiological importance of this active repression is demonstrated by the role of corepressors in acute promyelocytic leukemia whereby disruption of the corepressor complex or inhibition of histone deacetylase activity allows for redifferentiation and evidence of a hematological remission (29, 30, 31). In addition to their action in leukemia, the corepressors likely play a role in the action of hormone antagonists such as tamoxifen, which allow the estrogen receptor to recruit either NCoR or SMRT rather than coactivators (32, 33). Finally, the corepressors likely play a significant role in the manifestations of hypothyroidism. This is best demonstrated by the strong phenotypic differences between hypothyroid mice and mice who lack all TR (34). In addition, overexpression of an NCoR inhibitor in vivo reverses the gene expression abnormalities caused by hypothyroidism (35). What is not clear is whether the corepressors NCoR and SMRT serve overlapping functions or have separate actions based on what they in turn recruit and their tissue of expression (33).
The recruitment of NCoR and SMRT is mediated by the ligand-binding domain of nuclear receptors that interact with helical IDs in NCoR and SMRT centered around a IxxII sequence, termed the CoRNR box, which is essential for nuclear receptor binding (18). However, the structure of both the individual IDs in the corepressors and the ligand-binding domains of the NRs is different, which undoubtedly allows for the differential recruitment of corepressors. In addition, the presence of multiple corepressor IDs allows for a NR dimer to either recruit a single corepressor or multiple corepressors. Furthermore, corepressor recruitment is complicated by the DNA binding of NRs, which influences the ability of certain NRs to recruit corepressors. Data from a number of groups have established that homodimers of TR isoforms or the chimeric RAR oncoproteins found in acute promyelocytic leukemia (PML) favor the recruitment of corepressors (26, 27, 36). However, the stoichiometry appears to be different in each case. As demonstrated previously, a single NCoR molecule prefers to bind the TR homodimer, while the PML-RAR
dimer appears to recruit two SMRT molecules (36). Thus, the interaction of NRs with corepressor may require homodimer formation, suggesting a physiological role for the TR homodimer in gene silencing.
To address the mechanism of TR preference for the homodimer in this study, we used both in vivo and in vitro approaches. In mammalian cells, the TR homodimer was allowed to form in solution only in the presence of NCoR. Furthermore, this was most dependent upon the presence of intact N3 and N2 domains, indicating that each of these domains bound to an individual TR and functioned to bridge the homodimer. Furthermore, two NCoR IDs were required to allow the homodimer to form. In addition, similar experiments performed in the presence of SMRT IDs did not allow formation of the TR homodimer.
To more formally examine the mechanism underlying this stabilization, we examined the association and disassociation of the TR homodimer either in the presence or absence of the NCoR IDs. While the association of both the TR homodimer and the TR-NCoR complex was similar with the DR+4 element, their dissociation was markedly affected depending on the presence of NCoR. NCoR dramatically stabilized the TR complex on its response element and prevented its dissociation. Indeed, the function of NCoR on the TR is similar to that of RXR in the context of preventing dissociation of the TR from its target elements. However, RXR functions to decrease TR interactions with corepressors and decrease silencing (27). These data suggest that the TR homodimer may be held in place in the absence of ligand by NCoR to allow full recruitment of the multiprotein repression complex, especially on elements that favor its binding. Certainly, NCoR is only required for homodimerization in solution, but the presence of NCoR and potentially SMRT enhances homodimer binding to TREs and suggests that it may play an important role in silencing. Interestingly, the oncoproteins AML-ETO and PML-RAR
are required to dimerize to recruit corepressors to mediate their pathological effect, suggesting that this mechanism may be common among multiple transcription factors that recruit the corepressors (36, 37).
Alignment of the NCoR and SMRT discloses a number of significant differences in their IDs, consistent with their ability to differentially recruit separate nuclear receptors. N3, which is not present in SMRT, and N2 both possess a conserved isoleucine proximal to the beginning of the helical motif of LxxH/IIxxxI/L proposed by Perissi et al. (19) and confirmed in the recent crystallization of the antagonist-bound PPAR
LBD coupled to the S1 domain of SMRT (23). The importance of this isoleucine is especially significant for N3 given that it possesses an alanine instead of a leucine at position 3 (see Fig. 5
) of the helical motif. Indeed, when an alanine is introduced into either N2 or N1 in place of the leucine at position 3, NR binding is lost in a two-hybrid assay (19). Thus, the presence of an isoleucine at position 1 in N3 and a phenylalanine at position 6 suggests that they both allow for the formation of a proposed helix. Indeed, mutation of the phenylalanine disrupts NCoR binding, suggesting that its presence is essential. Furthermore, given that both N3 and N2 favor the TR, we hypothesized that the conserved isoleucine at position 1 also allows for specificity. Indeed, changing this isoleucine to an alanine in N3 caused a loss of all binding to the TR consistent with it being part of a novel helical domain present in N3. In addition, mutation of this residue to the corresponding valine found in S2 caused both N3 and N2 to lose binding to the TR both in EMSA and in mammalian cells, while changing the valine to an isoleucine in S2 enhanced TR recruitment. Finally, the presence of this isoleucine in N3 and N2 was required for corepressor function on a TRE in cells, consistent with its role in allowing for N3 and N2 to distinctly recognize the TR. Thus, given the critical role of N3 in mediating interactions with the TR, it is likely that an altered helical domain allows for specific binding to the TR ligand-binding domain when present in a homodimer. This is consistent with the differences seen between the TR LBD and PPAR
LBD in both the structure of the LBD and in the proposed amino acids that make contact with the corepressor helix (23, 28).
Thus, we propose that the TR homodimer is stabilized in the absence of ligand, on target response elements by the corepressor NCoR, which prevents dissociation of the TR. The specific recruitment of NCoR is mediated principally by a novel ID, N3, which possesses an altered helical structure that preferentially recognizes the TR homodimer and acts in concert with a more conserved corepressor ID, N2, which possesses a similar altered helical structure. Each of the corepressor IDs binds to an individual TR in the homodimer. This model implies that the recruitment of corepressors by NRs is distinct and mediated by difference both in the corepressor IDs and the NR LBDs.
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MATERIALS AND METHODS
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Plasmids and Transfections
All NCoR mutations made were carried using the QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA). The individual IDs and their mutants were cloned into PGEX-4T1 as EcoRI-XhoI fragments. As described previously, GST-N3 encompasses amino acids 19392021; N2, amino acids 20632142; and S2, amino acids 21192266 of SMRT (17). All mutations were confirmed by DNA sequencing. NCoRI and its mutants were inserted into either the Simian virus 40-driven expression vector PKCR2 or downstream of the Gal4-DNA binding domain in the Simian virus 40-driven expression vector pECE. VP16-TRß1 was constructed by inserting TRß1 downstream of the VP16 activation domain in the vector AASV. Gal4-TR contains the LBD of TRß1 downstream of the Gal4-DNA binding domain in pECE.
All transient transfections were performed in CV-1 cells, which were maintained as previously described (17). Transfections were performed using lipofectamine (Life Technologies, Inc., Gaithersburg, MD) such that each well contained equal amounts of plasmid DNA. In the mammalian two-hybrid experiments, each well received 800 ng of reporter and equal amounts of both the Gal4 and AASVP16 vectors. When NCoRI or its mutants were expressed in the two-hybrid assay, 100 ng were used per well. In experiments employing the DR4 reporter, 800 ng of reporter and 300 ng of TRß1 were used. pKCR2-NCoRI or its mutants (10 ng) were added to reverse repression. All wells received 10 ng of a cytomegalovirus ß-galactosidase expression vector to control for transfection efficiency. The cells were assayed for luciferase and ß-galactosidase activity 36 h after transfection. All experiments were performed in duplicate or triplicate, and luciferase expression was corrected for ß-galactosidase activity. The data are displayed as the mean ± SEM and represent pooled data from two to three separate experiments.
GST Fusion Proteins and EMSA
GST fusion proteins were expressed in DH5
or BL21 Escherichia coli expressing thioredoxin by induction with 0.1 mM isopropyl-ß-D-thiogalactopyranoside. The proteins were isolated as described previously and eluted from sepharose beads using a glutathione elution buffer. Verification of the integrity of protein synthesis was confirmed on SDS-PAGE. Protein quantification was performed using the Bradford assay.
EMSAs were carried out as previously described with a 32P-radiolabeled DR+4 probe (14), GST-corepressor proteins or in vitro translated NCoRI, and in vitro translated human TRß1. The integrity of all in vitro translated proteins was confirmed by the incorporation of 35S-methionine and direct visualization on SDS-PAGE. In each EMSA, 3 µl of in vitro translated TRß1 were used, and in EMSAs containing NCoRI or its mutants the amounts used were 1.5 µl or 3 µl. When GST-fusion proteins were used, the amount is indicated in the figure legend. Incubations were carried out with 100,000 cpm of probe for 20 min at room temperature. In experiments in which cold competitor probe was used, incubations were carried out for 20 min, and the indicated amounts (50-fold) of excess nonradiolabeled DR+4 were added. Incubations were continued for the indicated amount of time and then loaded to the running gel. Thus, the free probe and specific shifted complexes migrate at different sizes in these experiments. The nonspecific probe used as a competitor is the STAT3 binding site present in the murine TRH promoter (5'-CCACCAGGTTCCGGAAAGCGGGCGGGTCC-3') (38). Oligonucleotides were annealed and added in the indicated amounts. Densitometry was performed on EMSAs using NIH Image software. Lanes without competitor oligonucleotide were set at 100% in each experiment.
For antibody supershift experiments, reactions were carried out as described above for 20 min, at which time antibodies directed against TRß (Affinity BioReagents, Inc., Golden, CO) and NCoR (directed against a conserved C-terminal peptide in NCoR) were added for an additional 20 min before loading.
Nuclear Extract Preparation and Western Analysis
CV-1 cells were transiently transfected with equal amounts of pKCR2-NCoRI and its mutants and pKCR2 alone as well as pECE-Gal4-NCoRI and its mutants and pECE-Gal4 alone. Nuclear extracts were isolated following the procedure of Schreiber et al. (39), and equal amounts were loaded onto 10% SDS-PAGE. After electrophoresis the proteins were transferred overnight to nitrocellulose and probed overnight at 4 C with a 1:250 dilution of an affinity-purified anti-NCoR antiserum directed against a conserved C-terminal peptide in both human and murine NCoR. Western analysis was performed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).
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FOOTNOTES
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This work was supported by NIH Grants DK-56123 (to A.N.H.) and DK-02581 (to R.N.C.)
Abbreviations: ID, Interacting domain; LBD, ligand-binding domain; NCoR, nuclear receptor corepressor; NR, nuclear receptor; PML, promyelocytic leukemia; PPAR, peroxisomal proliferator-activated receptor; RAR, retinoic acid receptor; RXR, retinoic X receptor; SMRT, silencing mediator of retinoid and thyroid hormone receptors; STAT, signal transducer and activator of transcription; TR, thyroid hormone receptor; TRE, thyroid hormone response element.
Received for publication September 5, 2002.
Accepted for publication November 12, 2002.
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