Gene Silencing by Chicken Ovalbumin Upstream Promoter-Transcription Factor I (COUP-TFI) Is Mediated by Transcriptional Corepressors, Nuclear Receptor-Corepressor (N-CoR) and Silencing Mediator for Retinoic Acid Receptor and Thyroid Hormone Receptor (SMRT)

Hirotaka Shibata, Zafar Nawaz, Sophia Y. Tsai, Bert W. O’Malley and Ming-Jer Tsai

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors that belong to the steroid/thyroid hormone receptor (TR) superfamily and can repress the transcriptional activity of several target genes; however, the precise mechanism of this repression is unknown. Transfection of a Gal4 DNA-binding domain fused to the putative ligand-binding domain of COUP-TFI (Gal4-COUP-TFI) significantly represses the basal transcriptional activity of a reporter gene containing Gal4-binding sites. Cotransfection of COUP-TFI can relieve the Gal4-COUP-TFI repression in a dose-dependent manner. In contrast, COUP-TFI{Delta}35, which lacks the repressor domain (the C-terminal 35 amino acids), fails to relieve this repression. This finding suggests that the repressor domain of COUP-TFI may squelch a limiting amount of corepressor in HeLa cells. In addition, increasing concentrations of TRß also can relieve the COUP-TFI repression in a hormone-sensitive manner. Similarly, overexpression of increasing concentration of COUP-TFI, but not COUP-TFI{Delta}35, can squelch the silencing activity of the unliganded TRß. Collectively, these results indicate that COUP-TFI and TRß share a common corepressor(s) for their silencing activity. To determine which corepressor is involved in the COUP-TF-silencing activity, we used a yeast two-hybrid and in vitro GST pull-down assays to demonstrate that COUP-TFI can interact with the fragment of N-CoR (nuclear receptor-corepressor) encoding amino acids 921-2453 and the fragments of SMRT (silencing mediator for retinoic acid receptor and TR) encoding amino acids 29–564 and 565-1289, respectively. Interestingly, the fragment of SMRT encoding amino acids 1192–1495, which strongly interacts with TRß, interacts very weakly with COUP-TFI. Furthermore, overexpression of N-CoR or SMRT potentiates the silencing activity of COUP-TFI and can relieve the COUP-TFI-mediated squelching of Gal4-COUP-TFI activity. Therefore, our studies indicate that N-CoR and SMRT act as corepressors for the COUP-TFI silencing activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid/thyroid hormone receptors are ligand-dependent transcription factors that regulate diverse aspects of growth, development, and homeostasis by binding as monomer, homodimers, or heterodimers to their cognate DNA response elements to modulate transcription of target genes (1, 2, 3). Thyroid hormone (TR) and retinoic acid receptors (RAR) bind to cognate DNA response elements and repress basal promoter activity in the absence of ligand (3, 4, 5). The region important for repression may represent a unique interaction surface for contacting a particular target within the basal transcriptional machinery. Highly charged regions are a common feature of other repression motifs. These charged regions consist of basic residues (6) or acidic residues (7, 8). In contrast to these well-defined small repression motifs, the homologous repression regions of TR and RAR are large and appear to include multiple regions within the ligand-binding domains (LBDs) and hinge regions. Many interacting proteins have been identified and proposed to be important for mediating the repressor function. The identified molecular targets of repressors can be categorized into three groups (9): basal transcription factors, activators, and coregulators, such as coactivators and corepressors.

Silencing activity of unliganded TR and RAR requires limiting factors termed corepressors (5). Recently, two corepressors that bind to unliganded TR and RAR have been cloned using a yeast two-hybrid screen: N-CoR (nuclear receptor-corepressor) (10, 11) and SMRT (silencing mediator for RAR and TR) (12, 13, 14, 15). Binding of these corepressors is necessary for unliganded receptors to silence the activity of target promoters. Prevailing evidence suggests that binding of hormone changes the conformation of the receptors, which results in the release of the corepressor and recruitment of coactivators, thereby abolishing their silencing activity. These two corepressors exhibit significant sequence homologies in their receptor-interacting domains, suggesting the existence of a corepressor gene family. COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) belong to the steroid receptor superfamily and are classified as orphan receptors because their ligand has yet to be defined. There are two COUP-TF genes in mammals, COUP-TFI and COUP-TFII. COUP-TFs have been implicated in neurogenesis, organogenesis, and cell fate determination (16, 17, 18, 19). COUP-TFs can form stable homodimers and bind to a variety of hormone response elements recognized by other members of the steroid receptor superfamily, such as RAR, retinoid X receptor (RXR), TR, vitamin D receptor (VDR), peroxisome proliferator-activated receptor, and hepatocyte nuclear factor 4. COUP-TFs can thereby inhibit transcriptional activities of these receptors on both consensus and natural response elements (17, 20). Four mechanisms have been proposed to address COUP-TF’s ability to inhibit the transactivation function of other members of the steroid receptor superfamily. First, COUP-TFs repress the hormone-dependent transactivation of target genes by VDR, TR, and RAR through direct competition for occupancy of their response elements (21, 22, 23). Second, COUP-TFs heterodimerize with RXR to reduce the available concentration of RXR for heterodimerization with TR, VDR, RAR, and peroxisome proliferator-activated receptor and thus indirectly interfere with these receptors to transactivate their target genes (21, 22, 24, 25, 26, 27). Third, COUP-TFs can tether to DNA via LBD-LBD interactions with TR, RAR, and RXR to transrepress their activities (27). Finally, COUP-TFs have been shown to repress basal and activator-dependent transcriptional activities of various promoters when their binding site is placed upstream or downstream of these promoters (27). The silencing domain in COUP-TFs was localized to the C terminus of the putative LBD, which can be transferred to the heterologous Gal4 DNA-binding domain (DBD). Activators that can be repressed by COUP-TFs include acidic (Gal4-RII), glutamine-rich (Gal4-ftzQ), proline-rich (Gal4-CTF1P), and Ser/Thr-rich (Gal4-ZenST) transactivators (27). Because COUP-TF can repress such a diverse group of transactivators, it is unlikely that COUP-TF’s repression is through direct quenching of these transactivators or by interfering with their respective targets. It is rather likely that COUP-TFs interact with a common target, a putative corepressor(s) that mediates their repression.

To substantiate this hypothesis, we address two major questions in this paper; 1) Does COUP-TFI-mediated repression require corepressors? and 2) If it does, does COUP-TFI share corepressors with other receptors, such as TR and RAR? To answer these questions, we first examined whether cofactors are involved in COUP-TFI-mediated repression using either self-squelching or TR/COUP-TF-mediated mutual squelching experiments. Next, we examined whether COUP-TFI can interact with N-CoR or SMRT. Finally, we examined whether N-CoR or SMRT can function as a corepressor in COUP-TFI-mediated repression. Results from these experiments indicate that both N-CoR and SMRT can function as corepressors for COUP-TFI-mediated repression of target genes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
COUP-TFI-Mediated Transcriptional Repression Requires Corepressor(s)
The putative LBD of the COUP-TFI (amino acid 184–423) has been previously shown to contain a transcriptional repression domain. To repress transcription, it is generally believed that the COUP-TFI interacts with the basal transcriptional machinery either directly or indirectly through other cofactor molecules. To determine whether a cofactor(s) is required for this repressor activity, we carried out transfection experiments. Transfection of a Gal4 DBD fused to the COUP-TFI (amino acid 184–423) cDNA, designated as Gal4-COUP-TFI, significantly repressed basal promoter activity of the 17 mer x 4-thymidine kinase promoter-linked luciferase reporter DNA as shown previously (Fig. 1Go). This repression activity can be reversed by the exogenously expressed LBD of COUP-TFI (184–423), which does not bind to the reporter construct, in a dose-dependent manner. The observed release of repression cannot be attributed to the nonspecific effect of overexpression of COUP-TFI on Gal DNA-binding activity because overexpression of COUP-TFI had little effect on the basal promoter activity when cotransfected with the Gal4 DBD alone (27). In contrast, COUP-TFI mutant, COUP-TFI{Delta}35 (amino acid 184–388), which does not contain a repressor domain, cannot reverse Gal4-COUP-TFI-mediated repression. Overexpression of COUP-TFI or COUP-TFI{Delta}35 does not affect the basal promoter activity of Gal4 (data not shown). These results clearly indicate that a limiting factor, termed corepressor(s), is required for the activity of COUP-TFI-mediated repression. These results also suggest that the C-terminal 35 amino acids are required for interaction with the putative corepressor(s).



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Figure 1. COUP-TFI Is Able to Squelch the Repressor Activity of Gal4-COUP-TFI

HeLa cells were transfected with 0.1 µg Gal4-COUP-TFI cDNA, 0.3 µg 17 mer x 4-tk-luciferase reporter DNA, and increasing amounts of COUP-TFI or COUP-TFI{Delta}35 expression plasmid (0.2, 0.4, and 1.0 µg). Gal4 cDNA (0.1 µg) was transfected instead of Gal4-COUP-TFI in lane 1.

 
COUP-TFI Shares Common Corepressor(s) with TRß
To elucidate whether COUP-TFI shares common corepressors with TRß, squelching experiments were performed. Overexpression of TRß in the absence of T3 can reverse the basal repression activity of Gal4-COUP-TFI by squelching endogenous corepressors in a dose-dependent manner. In contrast, the liganded TRß cannot squelch the repression (lines 3–8, Fig. 2Go). Similarly, overexpression of TR mutant, TR168–456(V174A/D177A), which loses its silencing activity but retains hormone-dependent transactivation (28), cannot relieve the basal repression activity of Gal4-COUP-TFI (lines 9–11, Fig. 2Go). These findings strongly suggest that TRß- and COUP-TFI-mediated repression share the same corepressor(s).



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Figure 2. Unliganded TRß, but Not Liganded TRß or TR168–456 (V174A/D177A), Is Able to Squelch Repressor Activity of Gal4-COUP-TFI

HeLa cells were transfected with 0.1 µg Gal4-COUP-TFI cDNA, 0.3 µg 17 mer x 4-tk-luciferase reporter DNA, and increasing amounts of TRß or TR168–456 (V174A/D177A) expression plasmids (0.2, 0.5, and 1.0 µg). Cells were treated with 10-7 M T3 for 24 h in lanes 6–8. Gal4 cDNA (0.1 µg) was transfected instead of Gal4-COUP-TFI in lane 1.

 
In a reverse experiment, we examined whether COUP-TFI can squelch the repression activity of Gal4-TRß. As shown in Fig. 3Go, transfection of Gal4-TRß into HeLa cells significantly repressed the basal promoter activity of the reporter construct. Overexpression of COUP-TFI can reverse this repression in a dose-dependent manner. As expected, we observe no reversal of TRß-mediated repression by overexpression of the repression-defective COUP-TFI mutant, COUP-TFI{Delta}35. These results further suggest that common corepressors are shared by COUP-TFI and TRß for their repression activity.



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Figure 3. COUP-TFI, but Not COUP-TFI{Delta}35, Is Able to Squelch the Repressor Activity of Gal4-TRß

HeLa cells were transfected with 0.1 µg Gal4-TRß cDNA, 0.3 µg 17 mer x 4-tk-luciferase reporter DNA, and increasing amounts of COUP-TFI or COUP-TFI{Delta}35 (0.2, 0.4, and 1.0 µg). Gal4 cDNA (0.1 µg) was transfected instead of Gal4-COUP-TFI in lane 1.

 
Both N-CoR and SMRT Interact with COUP-TFI
Using the LBD of TRß as a bait in a yeast two-hybrid screen, the TR-interacting protein, SMRT, has been cloned (12, 14, 15). Similarly, Hörlein et al. (10) have cloned another TR-interacting protein, N-CoR, which has sequence similarity to SMRT. Both N-CoR and SMRT have been identified as corepressors for TR and RAR. We asked whether these two corepressors can function as corepressors for COUP-TFI. First, we examined whether COUP-TFI can interact specifically with these authentic corepressors. Using a yeast two-hybrid assay, we confirmed that these corepressors can interact with TRß in a hormone-dependent manner. As shown in Fig. 4Go, A and B, a fragment of N-CoR encoding amino acids 921-2453 and fragments of SMRT encoding amino acids 565-1495 and 1192–1495 can interact with TRß in a hormone-dependent manner as previously described (10, 12, 14). It should be pointed out that, to our surprise, a larger fragment of N-CoR encoding amino acids 190-2453, which contains a fragment of N-CoR encoding amino acids 921-2453, cannot interact with TRß in a yeast two-hybrid assay. Although the reason for this discrepancy is not clear at present, it is possible that the N-terminal repression domain of N-CoR encoding amino acids 190–920 is very potent and overrides the activation function of Gal4 in a yeast two-hybrid assay. This notion is further supported by our observation that a fragment of N-CoR encoding amino acids 190-2453 can interact with TRß in vitro (data not shown).



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Figure 4. Interaction of TRß or COUP-TFI with N-CoR or SMRT in Yeast Two-Hybrid Assays

A, Various fragments of SMRT and N-CoR constructs. The fragments of SMRT encoding amino acids 29–564, 565-1289, 565-1495, and 1192–1495 were constructed. The fragment of SMRT encoding amino acids 1192–1495 was obtained by yeast two-hybrid screen as a bait of the LBD of TRß. The fragments of N-CoR encoding amino acids 190-2453 and 921-2453 were constructed. In N-CoR, there are two repression domains (RD1 and RD2) as shown in the N-terminal region. The acidic-basic motif (AB) and the serine-glycine-rich domain (SG) in the central region are indicated. Glutamine-rich regions (Q) and predicted amphipathic {alpha}-helix (H) regions are also indicated. The most N-terminal region of SMRT (amino acids 1–483) contains four putative repeated motifs, and it has 44% identity with N-CoR. A series of deletion mutants of N-CoR or SMRT (illustrated in Fig. 4AGo) were created and tested for interaction in the yeast two-hybrid system with TRß (B) or COUP-TFI (C). For the experiments presented here, Gal4 DBD-TR168–456 or Gal4 DBD-COUP-TFI constructs were coexpressed with Gal4 AD-N-CoR or SMRT deletion mutants. Each two-hybrid pair was transformed into the yeast y190, the transformants were propagated, and the ß-galactosidase activity was determined. The effects of T3 on the TR/N-CoR or SMRT interaction in the presence of 1 µM T3 in the culture medium was also tested. Cotransformation of Gal4 DBD-COUP-TFI and Gal4 AD-TFIIB was used as a positive control. At least two independent yeast transformants were analyzed, and the results were determined as mean ± SEM.

 
Next, we examined the interaction between COUP-TFI and N-CoR or SMRT. We first demonstrated that no interaction between Gal4•DBD-COUP-TFI (amino acids 184–423) fusion and Gal4 activation domain (AD) (empty vector) was shown as a negative control (lane 1 in Fig. 4CGo). In contrast, as a positive control, we showed a strong interaction between Gal4•DBD-COUP-TFI and Gal4•AD-TFIIB (lane 7 in Fig. 4CGo). We then examined the interaction of various deletion fragments of N-CoR or SMRT with COUP-TFI (Fig. 4CGo). In a yeast two-hybrid assay system, the interaction of COUP-TFI with N-CoR or SMRT was detected when COUP-TFI was bound to DNA response element. In the absence of DNA response element, no interaction was observed (data not shown). A fragment of N-CoR encoding amino acids 921-2453 strongly interacts with COUP-TFI; however, a fragment of N-CoR encoding amino acids 190-2453 does not interact with COUP-TFI in a yeast two-hybrid assay. Similar to the TRß interaction study (Fig. 4BGo), the repression domains of N-CoR encoding amino acids 190–920 may interfere with the Gal4 activation domain function in a yeast two-hybrid assay; thus, a fragment of N-CoR encoding amino acids 190-2453 is not able to activate ß-galactosidase activity of the reporter. However, both N-CoR fragments encoding amino acids 190-2453 and 921-2453 were found to interact with COUP-TFI in an in vitro glutathione S-transferase (GST) pull-down assay because these in vitro interactions are presumably not influenced by the repressor function of N-CoR. Thus, a fragment of COUP-TFI encoding amino acids 56–423 can interact specifically with N-CoR in yeast and in vitro. On the other hand, fragments of SMRT encoding amino acids 29–564 and 565-1495 strongly interact with COUP-TFI (Fig. 4CGo). In contrast, a fragment of SMRT encoding amino acids 1192–1495, which interacts well with TR, interacts only weakly with COUP-TFI in a yeast two-hybrid assay (Fig. 4CGo). These results suggest that the COUP-TFI-interacting domain of SMRT is located mainly at amino acids 29–564 and 565-1191. These interaction data in yeast are further supported by the in vitro protein-protein interactions as shown in Fig. 5BGo. Both fragments of SMRT encoding amino acids 29–564 and 565-1289 are able to interact with GST-COUP-TFI, whereas a fragment of SMRT encoding amino acids 1192–1495 fails to interact with GST-COUP-TFI.



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Figure 5. Interaction between COUP-TFI and N-CoR or SMRT in in Vitro GST Pull-Down Assays

Radiolabeled N-CoR, SMRT, or TFIIB were tested for their abilities to bind to an immobilized GST-COUP-TFI fusion protein or to GST control protein. GST or GST-COUP-TFI fusion protein were expressed in yeast and purified by adsorption to a glutathione-Sepharose beads. A, The fragments of N-CoR encoding amino acids 190-2453 and 921-2453 and TFIIB, translated in vitro, were incubated with the GST control protein (lanes 2, 5, and 8) or GST-COUP-TFI (lanes 3, 6, and 9) for 30–60 min at 4 C with rocking. The GST or GST-COUP-TFI Sepharose beads were then extensively washed, and radiolabeled corepressor proteins (lanes 3 and 6) or TFIIB (lane 9) bound to the matrix were eluted with SDS loading buffer and analyzed by SDS-PAGE and autoradiography. Lanes 1, 4, and 7 represent each input protein. B, The fragments of SMRT encoding amino acids 29–564 (lanes 1–3), 565-1285 (lanes 4–6), and 1192–1495 (lanes 7–9) were radiolabeled and tested for their abilities to bind to an immobilized GST-COUP-TFI (lanes 3, 6, and 9 or to GST control protein (lanes 2, 5, and 8), while Lanes 1, 4, and 7 represent each input protein. The molecular weight of each protein is indicated.

 
N-CoR and SMRT Potentiate the Basal Repression by Gal4-COUP-TFI
From the above studies, we have shown that N-CoR and SMRT can interact with COUP-TFI specifically. We then asked whether these two corepressors are required to mediate COUP-TFI repression activity in mammalian cells. As shown in Fig. 6Go, neither N-CoR nor SMRT have a significant effect on transcription activity of the reporter construct in the absence of cotransfected Gal4-COUP-TFI. However, both N-CoR and SMRT can potentiate the repressor function of Gal4-COUP-TFI in a dose-dependent manner in HeLa cells. These data suggest that both N-CoR and SMRT can function as corepressors of COUP-TFI in HeLa cells. Thus, it is likely that they are the endogenous corepressors of COUP-TFI in HeLa cells.



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Figure 6. N-CoR and SMRT Can Potentiate Basal Repression Activity of Gal4-COUP-TFI

HeLa cells were transfected with 0.1 µg Gal4 or Gal4-COUP-TFI cDNA, 0.3 µg 17 mer x 4-tk-luciferase reporter DNA, and increasing amounts of N-CoR or SMRT expression plasmid (0.5 and 1.0 µg).

 
N-CoR and SMRT Can Reverse the Squelching of COUP-TFI-Mediated Repression
As shown earlier (Fig. 1Go), overexpression of COUP-TFI can squelch the repressor activity of Gal4-COUP-TFI. If N-CoR and SMRT are the limiting factors required for COUP-TFI-mediated repression, we expect that overexpression of N-CoR or SMRT should reverse this squelching activity. As shown in Fig. 7Go, overexpression of N-CoR or SMRT was able to relieve the squelching effects of COUP-TFI on Gal4-COUP-TFI repression function in a dose-dependent manner. On the other hand, overexpression of N-CoR or SMRT had no significant effect on transcription activity of the reporter construct in the absence of cotransfected Gal4-COUP-TFI (data not shown). Therefore, both N-CoR and SMRT can substitute for the endogenous corepressor(s) in mediating COUP-TFI’s repressor function.



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Figure 7. Reversal of COUP-TFI-Mediated Squelching of Basal Repression Activity of Gal4-COUP-TFI

HeLa cells were transfected with 0.1 µg Gal4 or Gal4-COUP-TFI cDNA, 0.3 µg 17 mer x 4-tk-luciferase reporter DNA, 1.0 µg COUP-TFI cDNA, and increasing amounts of N-CoR or SMRT expression plasmid (50, 100, and 200 ng).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown that both N-CoR and SMRT can function as corepressors for COUP-TFI-mediated gene silencing. Since COUP-TFI has been shown to interact with TFIIB in vitro (29, 30), it is proposed that COUP-TFI can interact with TFIIB in a nonproductive manner to interfere with the basal transcription. However, many coactivators and corepressors have been identified as positive and negative cofactors in the transcriptional control of target genes by members of the steroid/TR superfamily (15, 31). Therefore, it is tempting to speculate that some of the cofactors may also be involved in gene-silencing mechanisms of orphan receptors, such as COUP-TFI. Initial evidence for mediators of nuclear receptor transactivation came from the finding that transiently transfecting increasing amounts of an ER-expressing plasmid into cultured cells results in a bell-shaped curve of transcriptional activation and was attributed to the squelching of a coactivator(s) by the excessive amounts of estrogen receptor (32). By analogy with that, we demonstrated that a putative corepressor(s) may be involved in COUP-TFI-mediated basal repression because overexpression of COUP-TFI, but not COUP-TFI{Delta}35 (which has lost its repression domain), can squelch the repression activity of Gal4-COUP-TFI. Previously, we showed that a 15-amino-acid deletion from the C terminus (Gal4-COUP-TF{Delta}15) had little effect on Gal4-COUP-TF activity, whereas a 25-amino-acid (Gal4-COUP-TF{Delta}25) or a 35-amino-acid (Gal4-COUP-TF{Delta}35) deletion largely impaired its active repression function (27). Therefore, these data suggested that the C-terminal border of the major active repression function of COUP-TF is located within amino acids 408 and 398, and it is this region that may interact with the corepressor(s). In addition, the repressor domain of COUP-TFI may directly target the basal transcriptional machinery.

To examine whether a corepressor(s) can be shared between TRß and COUP-TFI, squelching experiments were performed. As shown in Fig. 2Go, unliganded but not liganded TRß can squelch the basal repression of Gal4-COUP-TFI. The evidence that the TR mutant, TR168–456 (V174A/D177A) (28), which does not interact with the putative corepressors, cannot squelch the basal repression of Gal4-COUP-TFI further supports the notion that common corepressors may be involved in basal repression between TRß and COUP-TFI. Furthermore, overexpression of COUP-TFI, but not COUP-TFI{Delta}35, can squelch the basal repression activity of Gal4-TRß, also suggesting that common corepressors can function for both COUP-TFI and TRß. Theoretically, the target of transcriptional interference may be either a basal transcription factor(s) or a corepressor(s). Transfection of COUP-TFI or TRß did not change the basal transcription of a PRE-tk-luciferase reporter lacking a proper response element (data not shown), suggesting that squelchers are unlikely to interfere directly with the function of any basal transcriptional machinery. Therefore, the target(s) of interference between COUP-TF and TRß is likely to be one or more transcriptional corepressor protein(s).

Because COUP-TFI can form homodimers by itself or form heterodimers with TRß (27), it is possible that the squelchers can form non-DNA-binding heterodimers with the repressors used in Figs. 1Go, 2Go, and 3Go (Gal4-COUP-TFI/COUP-TFI for Fig. 1Go, Gal4-COUP-TFI/TRß for Fig. 2Go, and Gal4-TRß/COUP-TFI for Fig. 3Go). If this is the case, inhibition of repressor activity may be due to inhibition of DNA binding rather than to squelching of a limiting corepressor(s). However, several lines of evidence suggest that this is not the case. First, both COUP-TFI and TRß have the ability to bind specifically to corepressors. Second, a mutant form of TRß (TR168–456 (V174A/D177A)), which loses its ability to interact with corepressor but retains its intact dimerization domain, is not able to inhibit the repression activity of Gal4-COUP-TFI. Third, COUP-TF mutant (COUP-TFI{Delta}35), which loses its ability in silencing, also loses its ability to squelch repressor activity of TRß. Finally and most importantly, we have demonstrated directly that both N-CoR and SMRT can mediate repressor activity of COUP-TFI (Fig. 6Go) and reverse the self-squelching activity (Fig. 7Go). Therefore, inhibition of repression activity by squelchers in Figs. 1Go, 2Go, and 3Go indeed results from squelching of a limiting corepressor(s).

Because squelching experiments showed that COUP-TFI may share a corepressor(s) with TRß, we examined protein-protein interaction between COUP-TFI and N-CoR or SMRT in a yeast two-hybrid assay and in in vitro GST pull-down assays as shown in Figs. 4Go and 5Go. The amino acid 921-2453 fragment of N-CoR interacts with COUP-TFI. This result is consistent with what has been observed in TR interaction studies (10, 33). Interaction between COUP-TFI and the amino acid 190-2453 fragment of N-CoR was not detected in a yeast two-hybrid assay, but was detected in an in vitro GST pull-down assay. This discrepancy is likely due to the potent repressor domain of the N-terminal portion of N-CoR, which suppresses the Gal4 activation function, resulting in interference with ß-galactosidase expression. Consistent with this interpretation, Seol et al. (33) also recently demonstrated that the repressor domain of N-CoR strongly interferes with the mammalian two-hybrid assay to study the protein-protein interaction between N-CoR and TR or RAR. On the other hand, the amino acid 29–564 and amino acid 565-1289, but not amino acid 1192–1495, fragments of SMRT strongly interact with COUP-TF in yeast and in vitro assays. Based on these data, we concluded that there are at least two COUP-TFI-interacting domains within the SMRT molecules. The lack of interaction between COUP-TFI and SMRT (1192–1495) is surprising because this region interacts very strongly with TRß. Thus, different repressors may interact differentially with SMRT. Finally, the corepressor-interacting region in COUP-TFI is localized in the extreme C terminus, which is quite different from the region of TR and RAR where hinge and N-terminal portions of LBD are involved in this interaction. Thus, repression function of COUP-TFs may act in a different way from that of TR and RAR (Fig. 8Go).



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Figure 8. Structure Domains of Repressors and Corepressors

A, Functional domains of COUP-TFI, TRß, and RevErb A. A/B, C, D, E/F are functional domains of receptors (3). CID (corepressor-interacting domain) is a sequence important for repressor function and contains a region that interacts with corepressors. The CoR box is a conserved region of human TR{alpha}, TRß, RAR{alpha}, and RevErb A. The CoR box of TRß is a repressor domain that interacts with corepressors; however, CIDs of RevErb A are different from the region of CoR box. B, Receptor-interacting domains of SMRT and N-CoR. Receptor-interacting domains of corepressors are indicated below each scheme of SMRT and N-CoR. In N-CoR, there are two repression domains (RD1 and RD2) as shown in the N-terminal region. The most N-terminal region of SMRT (amino acids 1–483) contains four putative repeated motifs and it has 44% identity with N-CoR. The acidic-basic motif (<1108>) and the serine-glycine-rich domain (SG) in the central region are indicated. Glutamine-rich regions (Q) and predicted amphipathic {alpha}-helix (H) regions are also indicated.

 
In support of this observation, N-CoR has been shown to function as a corepressor for RevErb A (34). However, a region homologous to the CoR box in the RevErb A, which is necessary for TR and RAR to interact with N-CoR, is not required for RevErb A-mediated repression. N-CoR contains two adjacent but distinct interaction domains, amino acids 2040–2239 and 2239–2296. The former fragment of N-CoR strongly interacts with both TR and RevErb A; however, the latter fragment of N-CoR interacts weakly and preferentially with RevErb A. Taken together, these data suggest that different nuclear receptors, utilizing different amino acid sequences, repress transcription by interacting with different region of N-CoR or SMRT as shown in Fig. 8Go.

Recently, an activator for COUP-TFII has been identified using a yeast two-hybrid screen, designated ORCA (orphan receptor coactivator) (35), and this factor is identical to a recently described ligand (p62) of tyrosine kinase- signaling molecule p56lck, suggesting that ORCA may link COUP-TFII and cell surface signal transduction pathways. This integrating role may be similar to what is observed with cAMP response element-binding protein CBP and the related protein p300. CBP plays a role in integrating cAMP second messenger and nuclear hormone receptor signal transduction pathways. Of interest, ORCA/p62 shares a small region of homology with CBP, suggesting a potential similarity in their mechanism of action. However, based on their data, ORCA/p62 does not bind directly to COUP-TFII-binding sites and COUP-TFII/ORCA complex is not detected in gel retardation assays. Therefore, it is possible that ORCA/p62 may function directly or indirectly by phosphorylating COUP-TFII or ORCA/p62 may overcome the function of a specific COUP-TFII-associated corepressor. Because ORCA/p62 can convert COUP-TFII into a transcriptional activator in a ligand-independent manner in mammalian cells, it is possible that COUP-TFI can also be activated by a coactivator(s) in a ligand-independent manner. Our preliminary data showed that overexpression of hSRC-1a (36), which is a general coactivator for members of steroid receptors, cannot relieve the repressor activity of Gal4-COUP-TFI in HeLa cells (data not shown).

In conclusion, we demonstrated that corepressors are involved in the mechanisms of COUP-TFI-mediated gene silencing, and that both N-CoR and SMRT can function as corepressors for COUP-TFI in mammalian cells. Therefore, orphan receptors such as COUP-TFI and RevErb A can function as a repressor in vivo by utilizing corepressors that are common for members of the TR and RAR subfamily.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mammalian Cell Culture, Transient Transfections, and Luciferase Assays
HeLa cells were routinely maintained in DMEM supplemented with 10% FBS. Twenty-four hours before transfection, 2 x 105 cells were plated per well on a six-well dish in DMEM containing 5% dextran-coated charcoal-stripped serum. Cells were transfected with the indicated DNAs using Lipofectin (Life Technologies, Grand Island, NY) according to the manufacturer’s guidelines. Usually, 1.8 µg of total DNA including 0.3 µg of reporter and 0.05 to 1.0 µg of expression vector were used per 35-mm diameter dish. Within 24 h, the DNA/Lipofectin mixture was removed and cells were fed with DMEM containing 5% stripped serum and the indicated hormones; cells were harvested 24 h later. Cells extracts were assayed for luciferase activity using the Luciferase Assay System (Promega, Madison, WI). Data are presented as an average of three separate experiments ± SEM. All transfections were performed at least three times in triplicate.

Cloning of SMRT by Yeast Two-Hybrid System
Yeast strains used are as follows. y190: MATa, leu2–3, 112, ura3–52, trp1–901, his2-D200, ade2–101, gal4{Delta}gal180{Delta}URA3 GAL-lacZ, LYS GAL-HIS3, cyhr. y187: MAT{alpha}, gal4, gal80, his3, trp1–901, ade2–101, ura3–52, leu2–3, -112, URA3 GAL-lacZ met-. BJ2168: MATa, prc1–407, Prb1–1122, pep4–3, leu2, trp1, ura 3–52. Yeast-selective media and plates were prepared according to Guthrie and Fink (37). The yeast strain y190 containing pAS1cyh2-TR168–456 was transformed with a human brain cDNA library in pGAD10 (Clontech, Palo Alto, CA) and plated on synthetic complete medium lacking tryptophan, leucine, and histidine (containing 25 mM 3-aminotriazole) as described by Durfee et al. (38). His+ colonies exhibiting ß-galactosidase activity using the filter lift assay were further characterized. ß-Galactosidase activity was determined using chlorophenol red ß-D galactopyranoside as described (38). To recover the library plasmids, total DNA from yeast was isolated and used to transform Escherichia coli (HB101) which lacks leu2 gene. Transformants were identified on minimal medium lacking leucine and containing ampicillin. To ensure that the correct cDNAs were identified, library plasmids isolated were retransformed into y190 containing pAS1cyh2-TR168–456, and ß-galactosidase activity was determined. The specificity of the interaction of cor 10.1 (SMRT 1192–1495), one of the 12 positive clones, with TR was determined by mating y190 containing pGAD10-cor 10.1 with the strain y187 containing either pAS1-SNF, pAS1-cdk2, pAS1-p53, or pAS1-lamin. The ß-galactosidase activity of these diploids was examined using the filter lift and chlorophenyl red ß-D galactopyranoside methods. The cor 10.1 clone was identical to recently identified corepressor SMRT. The yeast two-hybrid system was also used to determine protein-protein interaction between COUP-TFI and SMRT or N-CoR.

Protein-Protein Interaction by GST-Pulldown Assay
GST-COUP-TFI (pCBGST1-COUP-TFI) fusion protein was expressed and extracted in yeast strain BJ2168 as described previously (39). GST-pulldown assay was performed as described with modifications (4, 39): 30 µl glutathione-Sepharose beads stored in NENT buffer (500 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, 0.5% NP-40, 1 mM dithiothreitol, 6 mM MgCl2, and 8% glycerol) were incubated with yeast extracts containing GST-fusion proteins in a 1:1 vol ratio together with NENT buffer for 30–60 min at 4 C. Preparation of yeast extracts containing GST-fusion protein was described previously (39). Subsequently, the supernatant was removed and the beads were washed twice with 1 ml NENT buffer and twice with 1 ml transcription washing buffer (60 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, 0.05% NP-40, 1 mM dithiothreitol, 6 mM MgCl2, and 8% glycerol). In vitro-translated and -radiolabeled proteins were obtained using TNT Coupled Reticulocyte Lysate Systems (Promega). Five to 10 µl crude lysate were incubated with the beads in 200 µl transcription washing buffer for 2 h at 4 C. Finally, the beads were washed five times with 1 ml NENT buffer and proteins were solubilized in SDS loading buffer and analyzed on SDS-PAGE. The input lane contains 10% of the labeled protein used for binding.

Plasmids
Mammalian Expression Vectors
The expression plasmids pABgal147, pAB{Delta}gal, pABgalTRß, pAB{Delta}galTRß, pAB{Delta}gal-TR168–456 (V174A/D177A), pRSV-COUP-TFI, pRSV-COUP-TFI{Delta}35, and pRSVgalCOUP-TFI were described previously (5, 20, 21, 27, 28, 40). pCMX-N-CoR (10) and pCMX-SMRT (12) were generous gifts from Dr. A. J. Hörlein and Dr. J. D. Chen, respectively. The 17 mer x 4-tk-LUC reporter gene contains four copies of a 17-mer upstream activating sequence located upstream of the thymidine kinase promoter and luciferase gene. pCR3-SMRT565–1289 was constructed by TA Cloning (Invitrogen) of PCR-amplified product with primers 5'-AGCTGACGTCGACGCCTCGTG-3' and 5'-CTGCACCGCCTGGCTTCTAT-3' in which template cDNA was made by reverse transcription of human skeletal muscle mRNA (Clontech) with primer 5'-GCTGGCATGTTCCTGCACCG-3'. pCR3-SMRT565–1495 was constructed by inserting the EcoRI (filled)-BglII fragment of pGAD10-SMRT1192–1495 into the BglII-EcoRV site of pCR3-SMRT565–1289. pCR3-SMRT29–564 was constructed by TA Cloning (Invitrogen) of PCR-amplified product with primers 5'-AAGATTCCGAGCTCTGCTAC-3' and 5'-CACGAGGCGTCGACGTCAGC-3' in which template cDNA was made by reverse transcription of human skeletal muscle mRNA (Clontech) with primer 5'-GTGCGGGGACTTGGCGATCT-3'. pCR3-SMRT29–1495 was constructed by inserting the SalI fragment of pCR3-SMRT29–564 into the SalI site of pCR3-SMRT565–1495. pAB{Delta}galSMRT29–1495 was constructed by inserting the SalI (partial)-XbaI fragment into the PvuII site of pAB{Delta}gal. All PCR generates clones were sequenced to ensure that no mutation occurred during PCR reactions.

Yeast Vectors
The Gal4 DBD-TRß168–456 yeast expression plasmid (pAS1cyh2-TR168–456) was constructed by inserting the HindIII-SmaI blunt-ended fragment of pABgalTR168–456 into the SmaI site of pAS1cyh2 (38). The Gal4 DBD-COUP-TFI56–423 yeast expression plasmid (pAS1cyh2-COUP-TFI) was constructed by inserting the EcoRI-SmaI (filled) fragment of pGEM7Zf(+)-COUP-TFI into the SmaI site of pAS1cyh2. Yeast expression plasmid, pCBGST1-COUP-TFI56–423, was constructed by inserting the EcoRI-SmaI (filled) fragment (amino acids 56–423) of pGEM7Zf(+)-COUP-TFI into the SmaI site of pCBGST1 (39). Yeast expression plasmid, pACTII-N-CoR190–2453, was constructed by inserting the PvuI-SalI (filled) fragment of pCMX-N-CoR into the NcoI-XhoI site (filled) of pACTII (38). Yeast expression plasmid, pACTII-N-CoR921–2453, was constructed by inserting the HincII-SalI (filled) fragment of pCMX-N-CoR into the SmaI site of pACTII. pACTII-SMRT29–564 was constructed by inserting the SalI fragment (filled) of pCR3-SMRT29–564 into the NcoI (filled) site of pACTII. pACTII-SMRT565–1495 was constructed by inserting the SalI-XhoI fragment of pCR3-SMRT565–1495 into the NcoI (filled) site of pACTII. Yeast expression plasmid, pGAD10-SMRT1192–1495, was recovered from a yeast two-hybrid screen using pAS1cyh2-TR168–456 as a bait. Yeast expression plasmid, pACTII-TFIIB, was constructed by inserting the NcoI (partial)-EcoRI fragment of pGST-TFIIB (4) into the NcoI-EcoRI sites of pACTII.

In Vitro Transcription and Translation Vectors
pT7-N-CoR190–2453 was constructed by inserting the PvuI-SalI fragment of pCMX-N-CoR into the NcoI site (filled) of pT7ßSal (41). PT7-N-CoR921–2453 was constructed by inserting the HincII-SalI fragment of pCMX-N-CoR into the AccI site (filled) of pT7ßSal. pT7-SMRT29–564 was constructed by inserting the SalI fragment (filled) of pCR3-SMRT29–564 into the NcoI-EcoRI (filled) site of pT7ßSal. pT7-SMRT565–1289 was constructed by inserting the SalI-EcoRV fragment of pCR3-SMRT565–1289 into the NcoI-EcoRI (filled) site of pT7ßSal. PT7-SMRT1192–1495 was constructed by inserting the EcoRI fragment (filled) of pGAD10-SMRT1192–1495 into the HincII site of pT7ßSal. pT7-TFIIB was constructed by inserting the NcoI (partial)-EcoRI fragment (filled) of pGST-TFIIB (4) into the AccI site (filled) of pT7ßSal.


    ACKNOWLEDGMENTS
 
We thank members of our laboratories for critically reading this manuscript. We thank Drs. A. J. Hörlein and J. D. Chen for providing pCMX-N-CoR and pCMX-SMRT, respectively. We also thank Dr. Stephen Elledge for providing the yeast two-hybrid system.


    FOOTNOTES
 
Address requests for reprints to: Ming-Jer Tsai, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.

This work was supported by NIH Grants (DK-45641 to M.J.T. and HD-08188 to B.W.O.).

Received for publication January 15, 1997. Accepted for publication February 14, 1997.


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 DISCUSSION
 MATERIALS AND METHODS
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