Two Separate NCoR (Nuclear Receptor Corepressor) Interaction Domains Mediate Corepressor Action on Thyroid Hormone Response Elements

Ronald N. Cohen, Fredric E. Wondisford and Anthony N. Hollenberg

Thyroid Unit, Department of Medicine Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts 02215


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear corepressor (NCoR) binds to the thyroid hormone receptor (TR) in the absence of ligand. NCoR-TR interactions are mediated by two interaction domains in the C-terminal portion of NCoR. Binding of NCoR to TR results in ligand-independent repression on positive thyroid hormone response elements. The interactions between NCoR interaction domains and TR on DNA response elements, however, have not been well characterized. We have found that both interaction domains are capable of binding TR on thyroid hormone response elements. In addition, the NCoR interaction domains interact much more strongly with the TR than those present in the silencing mediator of retinoic acid and TRs (SMRT). Furthermore, deletion of either NCoR interaction domain does not significantly impair ligand-independent effects on positive or negative thyroid hormone response elements. Finally, both NCoR interaction domains appear to preferentially bind TR homodimer over TR-retinoid X receptor heterodimer in electrophoretic mobility shift assays. These data suggest that either NCoR interaction domain is capable of mediating the ligand-independent effects of TR on positive and negative thyroid hormone response elements.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The thyroid hormone receptor (TR), a member of the nuclear hormone receptor (NHR) superfamily, is a transcription factor that possesses transcriptional activity both in the presence and absence of its ligand, T3 (1). The TR mediates its ligand-independent and dependent actions by initially interacting with thyroid hormone response elements (TREs) either as a monomer, homodimer, or as a heterodimer with the retinoid X receptor (RXR) (2, 3, 4). Once bound to a TRE, the TR then interacts with accessory proteins and members of the basal machinery to mediate its transcriptional effects. In the absence of ligand, the TR is a potent silencer of gene expression when bound to certain positive TREs. This ligand-independent activity is mediated by a family of proteins termed nuclear corepressors. To date, two such family members have been identified NCoR/RIP13 (nuclear receptor corepressor) (5, 6) and SMRT (the silencing mediator of RARs and TRs) (7, 8). In the presence of ligand, TR releases corepressors and recruits coactivators such as SRC-1 (9, 10), TIF2 (GRIP1) (11), RIP 140 (12), CBP (13, 14), and p120 (15), to mediate ligand-dependent activation.

Full-length NCoR is a ubiquitously expressed 270 kDa protein (6). A second shorter isoform, RIP13a, of unknown significance also exists (16). NCoR is a modular protein and likely possesses four separate N-terminal repressing domains, two of which appear to interact with mSin3 and the histone deacetylase RPD3, HDAC1, to mediate repression (17, 18). It has been proposed that repression results from changes in chromatin structure mediated by this complex.

NCoR binds a portion of the TR hinge region termed the CoR box (6). Mutations in this region block the ability of unliganded TR to mediate repression. Two C-terminal NCoR domains, termed the interaction domains (ID I and ID II), appear to be involved in TR binding (16, 19). NCoR also appears to mediate repression by the orphan receptor RevErb (19). Protein interaction assays revealed that the NCoR interaction domains that mediate NCoR-RevErb binding are similar to those that mediate NCoR-TR effects; however, the domain of RevErb that binds NCoR is not homologous to the CoR box. The NCoR interaction domains have been identified using yeast two-hybrid and mammalian two-hybrid systems, but their functional significance on native TREs has not yet been defined. These interactions are critical as NCoR may be able to bind a number of NHRs in solution [peroxisome proliferator-activated receptors (PPARs) and RXR] but the presence of DNA response elements appears to functionally limit the interactions between NCoR and NHRs (20).

SMRT (TRAC2) is also a modular protein. It shares close to 40% amino-acid-identity with NCoR (21). It also contains N-terminal-repressing domains that appear to function through mSin3, and C-terminal interacting domains that interact with the TR, retinoic acid receptor (RAR), and RXR in a yeast two-hybrid system (7, 17). The interacting domains also share significant homology with those of NCoR. Recently, an endogenous inhibitor of SMRT, TRAC1, has been identified (8). It appears to be an alternatively spliced variant that contains the interacting domains but lacks the repressing domains.

We have identified a human homolog of NCoR that contains the C-terminal IDs, but not the N-terminal repressor domains (22). In transient transfection experiments, this construct inhibits endogenous NCoR function and has thus been termed NCoR inhibitor, or NCoRI. This truncated inhibitor of NCoR, which may or may not be expressed endogenously, provides an ideal tool to study the functional significance of the NCoR IDs on endogenous TREs. In this study, deletion constructs of hNCoRI were made that lack one or both of the known IDs. The functional characteristics of these constructs were assessed in transient transfection experiments using both positive (direct repeat, palindromic, and inverted palindromic) and negative (TRH) reporter elements. Electrophoretic mobility shift assays were also performed to characterize the binding of NCoRI deletion constructs to TR on DNA response elements and to compare structural binding with functional interactions.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Truncated Homolog of NCoR Serves as an Antirepressor
Human NCoR inhibitor (hNCoRI), a human homolog of NCoR, is an approximately 130 kDa protein, which was cloned using the yeast two-hybrid system (22). It has over 93% amino acid homology with the comparable region of mouse (m)NCoR (Fig. 1Go). It contains the C-terminal interacting domains of NCoR, but lacks the N-terminal-repressing domains (6). Recently, others have shown that a region present in NCoRI may, in fact, bind mSin3 and repress (18). However, we have demonstrated that hNCoRI has little repressing activity when linked to the Gal4 DNA-binding domain (data not shown). In transient transfection experiments, cotransfection of hNCoRI reverses ligand-independent repression on direct repeat (DR+4), palindromic (PAL), and inverted palindromic (LYS) reporters, presumably by interfering with endogenous NCoR function (22). To ensure that NCoRI possesses separate activity from the full-length moiety, we analyzed the function of NCoRI and NCoR on a positive and negative reporter in an identical transfection paradigm. As is shown in Fig. 2Go, NCoRI inhibits ligand-independent repression on the palindromic positive TRE, while full-length NCoR functions to enhance ligand-independent repression. In contrast, on the negatively regulated TRH reporter NCoRI is a strong activator of ligand-independent activation while NCoR has only a slight effect. However, the activation function on the negative reporter appears to be located downstream of the repressing domains as NCoRI is far more effective than NCoR. Thus, NCoRI is a potent antirepressor on both the positive and negative reporters. NCoRI has no effect on reporter gene activity in the absence of cotransfected TRß1 (data not shown).



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Figure 1. hNCoRI Deletion Constructs

A, mNCoR and hNCoRI are aligned schematically. Amino acid numbering is per mNCoR sequence. hNCoRI has 93% homology when compared with the comparable region of mNCoR. B, hNCoRI constructs lacking ID I and/or ID II were constructed, as described in Materials and Methods, and placed into the expression vector pKCR2. Amino acids corresponding to NCoRI sequence are identified with the corresponding sequence designations of mNCoR noted in parentheses.

 


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Figure 2. NCoR and NCoRI Have Separate Actions in CV-1 Cells

CV-1 cells were cotransfected with 1.7 µg of the indicated reporter construct and 80 ng of pKCR2-TR with either 330 ng of NCoR, NCoRI, or empty expression vector. The data are quantified as relative luciferase activity (mean ± SE) with basal activity in the absence of TR set as 1.

 
NCoR Prefers to Interact with the TR
The two C-terminal IDs of mNCoR, ID I and ID II, have been identified using yeast and mammalian two-hybrid systems (16) with the ligand-binding domain of the TR as bait. Similarly, two putative interacting domains have been identified in SMRT using solution interaction assays (23). It is unclear, however, what role these domains play on hormone response elements or in vivo. Alignment of the NCoR interacting domains with the SMRT amino acid sequence reveal considerable homology (53%) between NCoR ID I and an analogous region in SMRT and only 25% homology between NCoR ID II and the analogous region in SMRT (Fig. 3AGo). In contrast, the NCoR interacting domains are very well conserved between species as is shown in Fig. 3BGo.



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Figure 3. Sequence Alignments of the NCoR ID Sequences Differ from SMRT and Each Other, but Are Conserved across Species

A, The amino acid sequences of ID I and ID II from human NCoRI were aligned with the hSMRT AA sequence and the area of identity was shown. B, The ID I and ID II sequences from hNCoRI were aligned with the analogous sequences from mNCoR (6 ) and each other.

 
Given the differences in the amino acid sequences of the NCoR and SMRT interacting domains, we tested whether NCoR or SMRT preferred to interact with TRß1. We initially performed glutathione S-transferase (GST) pull-down assays and electrophoretic mobility shift assays (EMSA). In solution the ligand-binding domain of the TR appears to be able to interact equally well with both SMRT and NCoR (Fig. 4AGo); however, when equal amounts of the full-length protein are used in EMSA on a DR+4 element, NCoR is preferred by the TR, and little SMRT binds to either the TR homo or heterodimer (Fig. 4BGo). In addition, neither NCoR nor SMRT interacts with DNA in the absence of TR. These data suggest that either the DNA-binding region or other regions of the TR outside its ligand-binding domain alter the interaction between NCoR and SMRT. Further analysis of the interaction between the TR and NCoR and SMRT was performed in CV-1 cells. Using a mammalian two-hybrid system, where the ligand-binding domain of TRß1 was fused to the GAL4 DNA-binding domain and either corepressor to the VP-16 activation domain, we found that the interaction between TR and NCoR (Fig. 4CGo) was extremely strong and in fact stronger than the interaction between TR and RXR (data not shown). The interaction between TR and SMRT was much weaker in comparison. In contrast, Gal4-RAR{alpha} preferred to interact with SMRT rather than NCoR, ensuring that the VP-16 SMRT construct was adequately expressed and localized to the nucleus. Finally, we compared the ability of a dominant inhibitor of SMRT activity termed SMRTI (TRAC 1) (8) and NCoRI to function as antirepressors on the DR+4 reporter in CV-1 cells as shown in Fig. 4DGo. SMRTI was a consistently weaker inhibitor at all amounts transfected, in keeping with its poorer ability to interact with the TR. Unlike what has been reported for full-length mNCoR (24), hNCoRI acts as a potent antirepressor at all concentrations examined on the DR+4 reporter construct and other positive TREs (Fig. 4DGo).



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Figure 4. NCoR Prefers to Interact with the TR

A, GST-TR was used to pull down radiolabeled NCoR or SMRT. I, 20% input; G, GST alone. The concentration of T3 used was 10-6 M. B, EMSA was performed using in vitro translated proteins and a radiolabeled DR + 4 probe. The resolved complexes are indicated. C, CV-1 cells were transfected with 1.7 µg of the UAS reporter with 80 ng of the indicated GAL4 construct and 80 ng of the indicated VP-16 vector. The data are quantified as fold activation in the presence of empty VP-16 expression vector. D, CV-1 cells were transfected with increasing amounts of either NCoRI or SMRTI on the DR+4 reporter. The data are shown as fold repression in the presence of TR alone.

 
While both NCoR interacting domains appear to favor interactions with the TR, little is known about the ability of the individual NCoR IDs to interact with and function on endogenous TREs. Thus, deletion constructs of hNCoRI were made that lack ID I and/or ID II (Fig. 1Go) so that their function could be ascertained both functionally and structurally on endogenous TREs. Sequence alignment between these two regions in the hNCoRI protein disclosed only remote similarity (Fig. 3BGo).

Both IDs Reverse Ligand-Independent Repression on Positive TREs
To assess the function of the interacting domains on the ligand-independent activity of the TR, hNCoRI deletion constructs and TRß1 were transfected into CV-1 cells at a 1:1 DNA ratio. As shown in Fig. 5AGo, when cotransfected with two copies of the LYS element linked to the luciferase reporter, transfection of TRß1 causes approximately 3-fold repression of basal activity. Cotransfection of hNCoRI fully reverses this repression. Likewise, when a construct lacking ID I (hNCoRI {Delta}ID I) is cotransfected, there is also full reversal of ligand-independent repression. Similarly, hNCoRI {Delta}ID II reverses basal repression when cotransfected with TRß1. In contrast, hNCoRI {Delta}ID I/II, which lacks both IDs, does not reverse ligand-independent repression on the LYS element. Fig. 5BGo shows similar data, using the palindromic reporter. Again, hNCoRI reverses basal repression, as do constructs lacking either ID. In contrast to the LYS reporter, however, deletion of either ID I or ID II did impair somewhat the ability of the construct to reverse TR-mediated repression. In addition, the construct lacking both IDs again did not reverse repression.



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Figure 5. Both IDs Are Capable of Functionally Interacting with the TR on Positive TREs

CV-1 cells were cotransfected with 1.7 µg of LYS (A) PAL (B), or DR+4 (C) reporter plasmid and 80 ng of pKCR2-TR and/or 80 ng of pKCR2 and/or 80 ng of pKCR2-hNCoRI deletion construct. The data are quantified as fold repression (mean ± SE). D, CV-1 cells were transfected with GFP alone (mock) or the indicated NCoR construct and subjected to fluorescence microscopy.

 
On the DR+4 reporter (Fig. 5CGo), hNCoRI reverses basal repression, as do constructs lacking ID I or ID II. Unlike the PAL and LYS elements, however, hNCoRI {Delta}ID I/II is not fully deficient in its ability to reverse basal repression, as there is still 2-fold repression. Deletion of another 168 amino acids (hNCoR 1–302), which removes a portion of the mSin3-binding site (18) [mNCoR amino acids (AA) 1829–1940; hNCoRI AA 240–351], causes virtually all ligand-independent activity to be lost.

To rule out defective nuclear localization when IDs were deleted from NCoR, hNCoRI and the dual ID deletion construct were placed into a green fluorescent protein (GFP) expression vector and transfected into CV-1 cells. The cells were viewed under fluorescence microscopy 24 h after transfection to identify cellular localization of the fusion proteins. GFP vector alone exhibits diffuse staining in both the nuclear and cytoplasmic compartments. In contrast, hNCoRI {Delta}ID I/II-GFP was restricted to the nucleus (Fig. 5DGo), suggesting that lack of functional activity in transient transfection assays was not the result of impaired nuclear localization. In addition, hNCoRI-GFP also exhibited nuclear localization.

NCoR IDs Bind TRß1 on TREs and Preferentially Bind TR Homodimer
We next tested the ability of these hNCoRI constructs to interact with the TR on DNA response elements using EMSAs. As shown in Fig. 6AGo, wild-type TRß1 homodimer interacts strongly with hNCoRI. The addition of RXR{alpha} (or RXRß) causes NCoRI binding to be reduced without altering the position of the band. The addition of an RXR{alpha} antibody supershifts the TR-RXR heterodimer. The NCoR-TR complex is not supershifted by the anti-RXR{alpha} antibody, however, suggesting that this complex does not contain RXR. In contrast, an anti-TR antibody supershifts all complexes, including TR-NCoR (Fig. 6BGo). An antibody against C/EBP{alpha} does not shift any complex, indicating that these antibody supershift assays are specific. These data suggest that the NCoRI complex contains TR, but not RXR, and that NCoRI preferably binds to the TR homodimer over the TR-RXR heterodimer in this assay. Similar studies were performed using bacterially expressed NCoR-interacting domains and, even in the presence of limiting TRß1, the homodimer preferred to interact with NCoR (Fig. 6CGo). When the amount of GST-NCoR was increased by up to 50-fold (20 ng to 1000 ng) there was no difference found in the preference of binding.




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Figure 6. hNCoRI Preferentially Binds TR Homodimer

A gel mobility shift assay was carried out using in vitro translated proteins from rabbit reticulocyte lysate, except where indicated, and a labeled DR+4 oligonucleotide probe. After a 20-min room temperature incubation, 1 µl anti-RXR{alpha} (A), anti-TRb (B), or anti-C/EBP (B) antisera were added. Incubations were then carried out for an additional 20 min at room temperature. C, A gel-mobility shift assay was carried out using 20 ng of GST-NCoR with increasing amounts of TRß1 (shown as microliters of reticulocyte lysate) with the amount of RXR held constant in the presence of a labeled DR+4 oligonucleotide probe. D and E, CV-1 cells were transfected with the indicated constructs. The data are expressed as relative luciferase activity with the activity of GAL4-TR set as 1.

 
To further study the interactions between the TR and NCoR, we employed the mammalian two-hybrid assay. We first tested the ability of a GAL4-RXR{alpha} construct to affect both silencing by GAL4-TR and its interactions with NCoR. Cotransfection of GAL4-RXR with GAL4-TR will cause a heterodimer to form on the upstream activating sequence (UAS) response element in contrast to the TR homodimer when GAL4-TR is transfected alone. As is shown in Fig. 6DGo, cotransfected GAL4-RXR causes both a loss of silencing (GAL4-TR alone causes between 5- and 10-fold repression of basal activity) and diminishes considerably the ability of the TR to interact with the VP-16 NCoR construct. These results are consistent with the GAL4-TR homodimers avidly binding corepressor to mediate silencing. As the TR homodimer is shifted by NCoR in EMSA, we next investigated whether overexpressed NCoR could promote homodimerization of the TR in solution. Collingwood et al. (25) have previously shown that TR homodimers do not form in solution in the mammalian two-hybrid system. This is demonstrated in Fig. 6EGo as GAL4-TR is unable to interact with a VP-16-TR chimeric protein in CV-1 cells. We next asked whether increased amounts of NCoR could enhance the homodimer interaction. Indeed, cotransfected NCoRI allowed the TR homodimer to form in solution, suggesting that the homodimer may be stabilized by corepressor binding. In contrast, cotransfected SMRTI had no affect on TR homodimer formation, again supporting the preference of the TR for NCoR.

Since NCoRI appears to preferentially interact with the TR homodimer, the ability of the NCoRI deletion constructs to bind TRß1 on DNA response elements was assessed principally using the homodimer in EMSA. As shown in Fig. 7AGo, on the LYS element, TRß1 bound well as a homodimer. The addition of hNCoRI results in a supershift, representing TRß1-hNCoRI binding. Constructs lacking either ID alone bind TR on the LYS element, although these complexes migrate slightly further than TR-NCoRI, due to their smaller size. In contrast, constructs lacking both IDs do not bind TRß1 on the LYS element. However, on the PAL (Fig. 7BGo) and DR+4 (Fig. 7CGo) elements, although either interacting domain promoted TR binding, there appeared to be a preference for the ID II-containing construct. Constructs lacking either ID were unable to bind these elements.



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Figure 7. Both IDs Mediate Binding of NCoR to TR on DNA Response Elements

Gel mobility shift assays were carried out using 3–4 µl of in vitro translated proteins from rabbit reticulocyte lysate and 32P-radiolabeled LYS (A) PAL (B), or DR+4 (C) oligonucleotide probes. The labeled probe was incubated with in vitro translated TRb1 and hNCoRI deletion construct, or unprogrammed reticulocyte lysate as indicated.

 
To confirm that the presence of a heterodimer did not alter these results, EMSA was next performed on DR+4 in the context of RXR{alpha} as discussed above and shown in Fig. 8AGo. Similar results are seen with constructs lacking either ID I or ID II, suggesting that these IDs do not differentially bind TR-RXR vs. TR-TR on DNA response elements. Fig. 8BGo shows similar data in the context of 10 nM T3. As expected, there is loss of NCoR binding to TR in the presence of ligand, as well as a decrease in the homodimeric complex (26, 27). There is no significant binding of NCoRI to TR-RXR heterodimers in the presence of T3.



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Figure 8. NCoR IDs Bind TR Homodimer

The DR+4 probe was incubated with TRß1 and RXR{alpha} and/or hNCoRI deletion construct, in the absence (A) or presence (B) of 10 nM T3.

 
NCoR IDs Functionally Interact with TRß1 on Negative TREs by Enhancing Ligand-Independent Activation
To examine the function of NCoR IDs on genes negatively regulated by T3, constructs were cotransfected with the negatively regulated TRH promoter, linked to the luciferase reporter (28). In contrast to positive TREs, TR stimulates gene expression in the absence of T3 on negative TREs (28, 29, 30, 31). As seen in Fig. 9Go, cotransfection of hNCoRI with TRß1 resulted in an approximately 7- to 8-fold increase in ligand-independent activation. Cotransfection of hNCoRI {Delta} ID I or {Delta} ID II resulted in a similar stimulation of activity. In contrast, cotransfection of hNCoR {Delta} ID I/II only stimulated activity 2-fold above baseline. These data suggest that either ID is able to functionally interact with TR on negative TREs.



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Figure 9. hNCoRI Deletion Constructs Enhance Ligand-Independent Activation on a Negative TRE

TRH-pA3Luc (1.7 µg) was cotransfected with 80 ng pKCR2-TR and/or 80 ng pKCR2 alone and/or pKCR2-hNCoRI deletion construct. Each well was also transfected with 20 ng pCMV-ßGAL to control for transfection efficiency. The data are quantified as relative fold activation, where 1 represents activation in the presence of pKCR2-TR, but not pKCR2-hNCoRI, corrected for ß-galactosidase activity.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear corepressors silence transcription by interacting with unliganded TRs and RARs bound to their cognate response elements (5, 6, 7, 8). Once bound, corepressors silence through their ability to recruit RPD3 (HDAC1), which causes histone deacetylation of active chromatin and represses transcription (17, 18). Activation of transcription by ligand occurs through the reversal of repression (the release of corepressors), and the recruitment of coactivators, some of which contain endogenous histone acetylase activity (32, 33, 34). While coactivators appear to interact with the AF-2 domain of NRs on DNA through a leucine-rich motif (LxxLL) (35), it is unclear what allows the corepressor to bind to the CoR box of the TR in the absence of ligand. Furthermore, while SMRT and NCoR contain two separate TR IDs that have been previously been shown to function independently in solution (16, 19), they are dissimilar in sequence, and their function on native positive and negative TREs has not been addressed previous to this study.

We have initially compared the ability of the TR to interact with each of the corepressors. Our data demonstrate that the TR prefers to interact with NCoR on TREs and in mammalian cells. The EMSA data suggest that conformation on a DNA-response element may be important for this specificity. While others have shown interaction between SMRT and TR homodimers in EMSA, these studies were performed in the presence of significantly greater amounts of corepressor (20). The contrast between the GST-TR pull-down results, which showed equal binding to SMRT and NCoR, and the mammalian two-hybrid experiments, which showed an overwhelming preference by the TR for NCoR, is striking as both systems employed only the TRß1 ligand-binding domain. These experiments suggest that other nuclear proteins may influence the interactions between nuclear receptors and corepressors. Given the recent cloning of a novel corepressor, SunCoR (36), it is reasonable to hypothesize that other such proteins may influence the formation of corepressor complexes in cells. The preference of the TR for NCoR is supported by recent antibody microinjection studies, which demonstrate that inhibition of NCoR blocks silencing by the TR while anti-SMRT antibodies had no effect on TR-mediated silencing (37).

In transient transfection experiments, NCoR enhances the repressing function of TR. However, when increasing amounts of NCoR are transfected, paradoxical activation has been reported to occur with localization of transfected NCoR, but not endogenous NCoR, to nuclear dot structures (24). This complexity was addressed in the present study using NCoRI, which inhibits endogenous NCoR function, and thus provides an ideal tool for examining the function of the NCoR-interacting domains. Moreover, NCoRI progressively reversed ligand-independent repression on a positive TRE without any paradoxical effects on TR action. Furthermore, full-length NCoR did not activate when transfected in an identical paradigm.

When tested in cotransfection experiments, deletion of either interacting domain in the context of NCoRI had a negligible effect on the inhibitory activity of NCoRI on each of the three positive TREs tested. Furthermore, constructs containing a single ID retained the ability to bind to the TR on these TREs in EMSA, although ID II appeared to promote a stronger interaction. Comparison of the NCoR- and SMRT-interacting domains shows that ID I is quite similar while ID II is more dissimilar. Thus, the increased avidity of ID II for the TR is consistent with the preference of NCoR for the TR. Functional activity of NCoRI was abolished on the PAL and LYS reporters when both IDs were deleted. This NCoRI construct (hNCoRI {Delta}ID I/II) localized to the nucleus when fused to GFP, implying that this functional defect was not due to defective nuclear transport. When examined in EMSA, hNCoRI {Delta}ID I/II was unable to bind to TR on any of the response elements studied. Therefore, the lack of functional activity of this construct on the PAL and LYS elements results from its inability to bind to the TR on DNA. In contrast, hNCoRI {Delta}ID I/II retained a small but consistent inhibitory function on the DR+4 reporter without evidence of DNA binding, raising the possibility that the region remaining of NCoR may interact with TR to mediate repression on DR+4, or that the mSin3-binding site present in this construct may be important in mediating repression on this TRE. In fact, deletion of a portion of the mSin3 ID resulted in further loss of NCoRI function on the DR+4 element.

In contrast to ligand-independent repression on positive TREs, the TR causes ligand-independent activation in tissue culture on negative TREs. While the mechanism underlying negative regulation has not been defined, it is clear that TR-binding sites exist in the regulatory regions of genes negatively regulated by the TR (28). We have previously demonstrated that cotransfected NCoRI enhances ligand-independent activation on the TRH promoter in the presence of TRß1 and TR{alpha}1 (22). Deletion of either interacting domain does not impair the ability of hNCoRI to activate the TRH promoter. However, once both interacting domains are deleted, enhancement of ligand-independent activation is all but lost. Interestingly full-length NCoR also slightly activated the TRH promoter, as has been reported previously (38), but the effect was far less than that seen with NCoRI. These differences may be the result of the different cell types used as well as the amounts of cotransfected NCoR or NCoRI. Further studies will be needed to define the mechanism by which the corepressor influences negative regulation. The data presented here indicate that a direct interaction between the TR and NCoR is needed to enhance ligand-independent activity.

In the present study, we have also addressed the ability of hNCoRI to interact with either the TR homodimer of TR/RXR heterodimer. It is unclear in vivo what is the preferred TR configuration in the absence of ligand. Previous work by Zamir et al. (20) has demonstrated that the TR homodimer and TR/RXR heterodimer can both bind nuclear corepressor on a DR+4 element (20). These studies did not address the relative preference of homo- or heterodimer for the corepressors. In addition, it is also clear from another study (39) that RXR/TR{alpha}1 heterodimers can interact in cells with NCoR. The data presented here also show an interaction between NCoR and RXR/TRß1 heterodimers in the mammalian two-hybrid assay. However, this interaction is considerably weaker than the interaction with TR homodimers. Furthermore, in the mammalian two-hybrid system, RXR-TR heterodimers also silence less well. In addition, NCoRI appears to allow the TR homodimer to form in solution through its interacting domains. Further support for the preference of the homodimer for NCoR comes from gel-mobility shift assays on TREs, which demonstrate that the TRß1 homodimer binds in vitro translated NCoR and bacterially expressed NCoR, while the heterodimer demonstrates little avidity for NCoR. These data support the concept that the homodimer may be able to play a role in thyroid hormone action through its strong interaction with nuclear corepressors. While our data do not rule out that the heterodimer is an active silencer, they suggest that the homodimer preferentially interacts with each of the NCoR interacting domains. An alternative explanation could be that the gel-mobility shift assay is too insensitive to detect in vivo interactions between the TR/RXR heterodimer and NCoR and that interaction studies are cell specific. Further work will be necessary to clearly define the role of the in vivo configuration of TR when ligand-independent repression occurs and to determine the exact stoichiometry of the homodimer and heterodimer interactions.

In summary, we have demonstrated that the two separate NCoR domains interact with the TR on both positive and negative TREs. Furthermore, individual IDs can mediate dominant inhibition of endogenous NCoR activity, suggesting that the separate IDs are powerful mediators of ligand-independent interaction with the TR. The IDs are remarkably conserved across species; however, the two IDs themselves share only remote homology with each other. While the two IDs may be present to ensure strong interactions with TR, our data suggest that either ID alone allows NCoR-TR interactions to occur on native TREs. Further work on these modular domains will help delineate the specific amino acids necessary for ligand-independent interactions with the TR and will help identify the important differences that allow the TR to interact much more strongly with NCoR. This may allow for the identification of other such proteins that can interact with the TR to mediate its ligand-independent functions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The positive TRE constructs each consist of two copies of DR+4 palindromic, or F2LYS elements upstream of the TK109 promoter in the vector pA3Luc (22, 40). The human TRH promoter from -900 to +55 was also cloned into pA3Luc (28). Human TRß1 was cloned into the SV40-driven expression vector pKCR2, as previously described (40). A 868-AA NCoR fragment, termed hNCoRI, was cloned into pKCR2 as previously described (22). Full-length NCoR (a gift of C. Glass) was cloned as a NotI fragment into pKCR2. SMRTI was cloned as a SalI–EcoRI fragment of SMRT (a gift of R. Evans) into pKCR2. SMRTI was also cloned into AASVP-16 (a gift of S. Weissman), using PCR from full-length SMRT, to encompass AA 657-1495. The NCoRI construct lacking ID I was made by deleting AA 625–701 and creating an in-frame stop codon with a HindIII deletion. The construct lacking both ID I and ID II was made by PCR amplification of a portion of NCoRI from AA 1–470 inserted next to the novel stop codon created by the HindIII deletion. The construct lacking ID II was created by PCR amplification of AA 1–464 and AA 557–868 joined by a novel Sac-1 site created next to AA 464. NCoR 1–302 was obtained by a BglII deletion of the C-terminal portion of NCoRI in pKCR2. AASVP16-NCoRI was created by ligating AA 215–868 of NCoRI into AASVP16. The Gal4 and UAS-TKLuc constructs have been described previously (15). Gal4-TR was created by ligating a PstI–EcoRI fragment from hTRß1 encompassing AA 203–461 into the Gal4 vector. GAL4 RXR{alpha} encompasses AA 201–462 of human RXR{alpha} in frame with the GAL4 DNA-binding domain. GST-TR and AASV-TR were created by ligating the ligand-binding domain of the TR from Gal4-TR in frame with the GST moiety in PGEX4T1 and the VP-16 moiety in AASV, respectively. GST-NCoR was created by ligating the two interacting domains of hNCoR (AA 2063–2300) into PGEX 4T1. Constructs were cloned into pKCR2 for transient transfection experiments and pEGFP (CLONTECH, Palo Alto, CA) for cellular localization studies. The integrity of each plasmid was confirmed by restriction endonuclease digestion and dideoxy sequencing. All plasmids for transfection were purified using column (Qiagen, Chatsworth, CA) purification.

Cell Culture and Transient Transfection
CV-1 cells were maintained in DMEM supplemented with L-glutamine, 10% FCS, 100 µg/ml penicillin, 0.25 µg/ml streptomycin, and amphotericin.

Transient transfections using NCoRI alone were performed using the calcium phosphate technique in six-well plates with each well receiving 1.7 µg of reporter and 80 ng of pKCR2-TR and/or equal amounts of pKCR2 alone and/or equal amounts of pKCR2-NCoRI construct, except as noted. Each well also received 20 ng of a ß-galactosidase expression vector to control for transfection efficiency. Studies involving the full-length murine NCoR included 330 ng of expression vector per well. Fifteen to 18 h after transfection, the cells were washed in PBS, and refed with DMEM with 10% steroid hormone-depleted FBS. To remove steroid and thyroid hormones, FBS was treated for 24 h at 4 C with 50 mg/ml activated charcoal (Sigma Chemical Co., St. Louis, MO) and 30 mg/ml anion exchange resin (type AGX-8, analytical grade, Bio-Rad, Richmond, CA). Forty to 44 h after transfection, the cells were harvested in extraction buffer and assayed for both luciferase and ß-galactosidase (Tropix, Bedford, MA) activity. Experiments were performed in triplicate and repeated two to three times. The data are the pooled results ± SEM.

Transient transfections to assess cellular localization of hNCoR constructs were performed in 100-mm tissue culture dishes. Twenty micrograms of pEGFP-hNCoRI construct or pEGFP alone were transfected using the calcium phosphate technique. Twenty four hours later, cells were washed in PBS and refed with DMEM. Cells were then visualized under fluorescence microscopy.

EMSAs
Gel-mobility shift assays were performed with proteins derived either from a coupled in vitro translation/transcription reaction in reticulocyte lysate (Promega, Madison WI) or from Escherichia coli as GST-fusion proteins. Either TRß1 or RXR{alpha} (0.5 to 4 µl) with equal amounts of NCoRI or SMRTI construct were used, as well as 50,000–150,000 cpm of a 32P-radiolabeled DR+4 (5'-AGGTCACAGGAGGTCA-3'), palindromic (5'-AGGTCATGACCT-3'), F2-LYS (5'-TGACCCCAGCTGAGGTCA-3'), oligonucleotide probe. When employed, 20–1000 ng of GST-NCoR were used. Incubations were carried out at room temperature, and complexes were resolved on 5% nondenaturing acrylamide gels, followed by autoradiography. The concentration of T3 used was 10 nM. EMSA with antibody supershifts (anti-RXR{alpha}, Santa Cruz Biotechnology, Santa Cruz, CA; anti-TR, Affinity Bioreagents, Inc., Golden, CO; anti-C/EBP, Santa Cruz Biotechnology) were carried out similarly, except that in vitro translated proteins and probe were incubated at room temperature for 20 min, followed by addition of 1 µl antibody. The resulting complexes were incubated for another 20 min at room temperature and then resolved on a 5% nondenaturing gel, followed by autoradiography.

GST-Protein Binding Assays
GST-TR was expressed in BL21 E. coli expressing thioredoxin (41) by induction with 0.1 MM isopropylthio-ß-D-galactosidase (IPTG) at 30 C. GST-NCoR was expressed in DH5{alpha} E. coli under similar conditions. The proteins were isolated by lysis with lysozyme and purified on Sepharose beads. Verification of protein synthesis was obtained on SDS-PAGE. GST-TR (50 µl) and equivalent amounts of GST alone were incubated with 6 µl of [35S]methionine-labeled in vitro translated NCoRI or SMRTI. The concentration of T3 used was 10-6 M. After extensive washing, the bound proteins were eluted by boiling in SDS-PAGE loading buffer and run on SDS-PAGE.


    ACKNOWLEDGMENTS
 
We would like to thank R. Evans, C. Glass, and N. Moghal for plasmids and A. Takeshita for the thioredoxin-expressing BL21 E. coli.


    FOOTNOTES
 
Address requests for reprints to: Dr. Anthony Hollenberg, Thyroid Unit, Department of Medicine, Beth Israel-Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215. E-mail: thollenb{at}bidmc.harvard.edu

This work was supported by NIH Grants to A.N.H. (5DK 02354) and F.E.W. (3DK49126).

Received for publication April 28, 1998. Revision received June 9, 1998. Accepted for publication July 10, 1998.


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 RESULTS
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 MATERIALS AND METHODS
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