Silencing Subdomains of v-ErbA Interact Cooperatively with Corepressors: Involvement of Helices 5/6

Kerstin Busch, Bernd Martin1, Aria Baniahmad, Joseph A. Martial, Rainer Renkawitz and Marc Muller

Laboratoire de Biologie Moléculaire et de Génie Génétique (K.B., B.M., J.A.M., M.M.) Institut de Chimie-B6 Université de Liège B-4000 Liège, Belgium
Genetisches Institut (K.B., A.B., R.R.) Justus-Liebig-Universität D-35392 Giessen, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the thyroid hormone receptor (TR) family act on vertebrate development and homeostasis by activating or repressing transcription of specific target genes in a ligand-dependent way. Repression by TR in the absence of ligand is mediated by an active silencing mechanism. The oncogene v-ErbA is a variant form of TR unable to bind hormone and thus acts as a constitutive repressor. Functional studies and mutation analysis revealed that the TR/v-ErbA silencing domain is composed of three silencing subdomains (SSD1–3) which, although nonfunctional individually, synergize such that silencing activity is restored when they are combined in a heteromeric complex. Here we demonstrate, using protein interaction assays in vitro and in vivo, that the inactive v-ErbA point mutant L489R within helix 5/6 in SSD2 fails to interact with the two corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptor). Furthermore, mutants in SSD1 and SSD3 exhibit a reduced corepressor recruitment corresponding to their weak residual silencing activity. In mammalian two-hybrid assays, only the combination of all three silencing subdomains, SSD1–3, leads to a cooperative binding to the corepressors N-CoR or SMRT comparable to that of the full-length v-ErbA repression domain. In conclusion, full silencing activity requires corepressor interaction with all three silencing subdomains, SSD1–3. Among these, SSD2 is a new target for N-CoR and SMRT and is essential for corepressor binding and function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional regulation by steroid/thyroid hormones and retinoids is a critical component in controlling many aspects of animal development, reproduction, and metabolism (1, 2, 3, 4). The functions of these hormones are mediated by intracellular receptors, which comprise a large superfamily of ligand-dependent transcription factors (2, 4). The thyroid hormone receptor (TR) is bound to its cognate DNA sequence upstream of specific target genes (5) and activates transcription in the presence of its ligand, but represses transcription in the absence of hormone (6, 7). Repression by TR is mediated by its C-terminal ligand binding domain (LBD) (8, 9), which is known to harbor additional functions including ligand binding, transcriptional activation, nuclear localization, and dimerization (1, 10). The LBD represents a separable silencing domain that confers a true silencing activity (11, 12) when fused to the heterologous DNA-binding domain (DBD) of the yeast transcription factor Gal4 (8). A similar silencing function is found in the oncogene v-ErbA, a viral derivative of the chicken TR{alpha}, which is unable to bind hormone and functions as a constitutive repressor of target genes (6, 13). V-ErbA lacks the activation domain AF-2-AD/{tau}C/{tau}4 at the C-terminal end (14, 15, 16). Deletion or mutation of this domain converts TR or retinoic acid receptor (RAR) into potent constitutive transcriptional repressors (9, 17, 18, 19). V-ErbA and constitutively repressing TR or RAR mutants were shown to cause defects in cell differentiation and development (20, 21, 22, 23, 24, 25, 26, 27, 28). Thus, transcriptional repression by unliganded receptors appears to play an important role in regulating cell growth and differentiation.

The molecular mechanisms of the repression by nuclear hormone receptors have been the object of intense research. Interactions of the silencing domain of TR with factors of the basal transcription machinery, such as transcription factor (TF)IIB, TATA-binding protein, and TBP-associated factor (TAF) 110, have been shown in vitro (29, 30, 31, 32, 33, 34). Also, transcriptional regulation was shown to involve additional cofactors (reviewed in Refs. 35, 36, 37). In the absence of hormone, interaction of the TR with the corepressors N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) (38, 39, 40, 41, 42, 43) leads to subsequent recruitment of a Sin3/histone deacetylase complex able to change nucleosomes into a repressive state. In the presence of hormone, a conformational change involving helix 12 in the AF-2-AD/{tau}C/{tau}4 activation domain of TR or RAR (9, 44, 45, 46) induces the release of corepressors and the binding of coactivators. Protein interaction studies identified the N-CoR-box within the hinge region (38, 39) and the ninth heptad repeat in helix 11 (47, 48) of TR as direct interaction regions with these corepressors.

We previously described that the repression domain of TR and v-ErbA consists of three different subdomains (SSD1, SSD2, and SSD3; see Fig. 1Go), which show no silencing activity alone but which synergize in repression when they are combined (49, 50). The molecular mechanism for this functional reconstitution of the three subdomains is still unknown. The structural organization of the v-ErbA silencing domain in three subdomains is further illustrated by the positions of different point mutations that affect the repression function (50). Mutants located in SSD1 (P396R and P398R) and in SSD3 (L544R) present a severely weakened activity and mutants in SSD2 (P481R and L489R) were completely inactive. In a first attempt to investigate the involvement of limiting cofactors in the silencing activity, we performed in vivo titration (squelching) experiments (50). Expression of the chimeric effector protein Gal-ErbA and of large amounts of the v-ErbA silencing domain as competitor resulted in relief of silencing on the pUAS-tk-CAT reporter plasmid. Overexpression of mutants mediating wild-type repression activity also alleviates the repression. In sharp contrast, none of the inactive mutants was able to compete for silencing cofactors.



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Figure 1. Point Mutants within the Three Silencing Subdomains SSD1–3 of the V-ErbA Repression Domain

The v-ErbA C-terminal repression domain is shown and the three silencing subdomains SSD1 (aa 362–434), SSD2 (aa 434–508), and SSD3 (508–639) (50 ) are indicated. The position of several functionally relevant regions are shown: the hinge region including the N-CoR-box (38 ) in SSD1, helix 5/6 in SSD2, and helix 8 and the ninth heptad repeat/helix 11 (47 48 ) in SSD3. Three types of point mutants have been used in this work: P475R in SSD2 and I537R and L540R in SSD3 display wild-type repression activity (+); P396R and P398R in SSD1 and L544R in SSD3 exhibit a weakened silencing function (+/-); and mutant L489R in SSD2 is fully inactive (-).

 
Here, we directly investigate the protein-protein interaction of the wild-type and mutant v-ErbA silencing domain or the three silencing subdomains with the two corepressors N-CoR or SMRT in vitro and in vivo. We show that the silencing activity of several mutants of v-ErbA correlates with their capacity to recruit the corepressors N-CoR and SMRT. We show that the combination of the three subdomains results in a cooperative association with the corepressors, presumably leading to the reconstitution of the full repression. Furthermore, our results indicate that helices 5/6 (44) in SSD2 play a crucial role in these interactions and thus constitute a new direct interaction interface for corepressors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutants in SSD2 Cannot Be Complemented in Silencing by Mutants in Other Subdomains
Our previous results showed that none of the silencing-deficient v-ErbA mutants (Fig. 1Go) were able to compete with wild-type v-ErbA for silencing cofactors in vivo (50). A simple model for corepressor interaction with v-ErbA silencing subdomains would predict that inactive mutants in different subdomains should be able to complement each other. Therefore, we performed complementation experiments by combining repression-defective v-ErbA mutants in each of the subdomains. For this purpose, we used the well established Gal4-DBD-fusion system, in which amino acids 1–147 of Gal4 are sufficient to mediate nuclear translocation, dimerization, and specific binding to the upstream activator sequence (UAS), whereas the transactivation functions are deleted. In particular, we previously showed that two different Gal4 fusion proteins are able to heterodimerize via this domain (49). We cotransfected combinations of expression plasmids for different Gal-ErbA mutants, defective in silencing, with the reporter plasmid p(UAS)x4-tk-CAT in L-tk- cells. The wild-type Gal-ErbA and the indicated mutants are expressed at similar levels in mammalian cells (Ref. 50 ; see also Fig. 3Go). Three different point mutants within each of the previously defined silencing subdomains SSD1–3 were used.



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Figure 3. V-ErbA/SMRT Interaction in Vitro Requires SSD2 and Is Weakened by Mutations in Other Subdomains

EMSA analysis was performed to analyze SMRT interactions with wild-type and mutant v-ErbA. Extracts from COS-1 cells transfected with the indicated expression plasmids were incubated with bacterially expressed and purified nuclear receptor interaction domain of SMRT (C.SMRT aa 982-1495) fused to GST (600 ng) and tested with the UAS sequence as a probe. Extracts from cells transfected with pGal4-DBD or the empty expression vector {delta} gal (C) with or without GST.C.SMRT are shown as controls. The complex obtained with the Gal-ErbA fusions (lower arrow) and the supershift with GST.C.SMRT are indicated (upper arrow).

 
Expression of the Gal4 DBD alone did not affect the activity of the p(UAS)x4-tk-CAT reporter gene, while expression of the wild-type Gal-ErbA resulted in 18-fold repression (Fig. 2BGo). As expected, the two mutant Gal-ErbA in SSD1 and SSD3 (P398R and L544R) displayed reduced silencing activity (2-fold) while mutant L489R in SSD2 was completely inactive. Coexpression of the mutant P398R in SSD1 and L544R in SSD3 restored 10-fold repression activity. In contrast, the completely silencing-deficient mutant within SSD2 (L489R) could not be complemented by coexpression of either mutant L544R or mutant P398R. Similar results were obtained when other mutants in each of the silencing subdomains were used (not shown). The synergistic effect observed when two point mutants, respectively, in SSD1 and SSD3 are coexpressed indicates that the inactivating mutation of only one silencing subdomain can be complemented by a mutant in the other subdomain. However, the mutation in the central subdomain SSD2 cannot be rescued by coexpression of any of the mutants in other subdomains.



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Figure 2. The SSD2 Mutant Cannot Be Complemented by Mutants in SSD1 or SSD3

A, Schematic representation of wild-type Gal-erbA and indication of the position of the mutations in the different silencing subdomains. B, Cotransfection of the reporter plasmid p(UAS)x4-tk-CAT with expression vectors for the wild-type or mutant Gal-ErbA fusion proteins was performed into L-tk- cells, as indicated. In control experiments, a vector coding for the Gal4-DBD was added to keep the amount of expression plasmid constant. The graph represents fold repression obtained relative to Gal4-DBD alone. C, Cotransfection of p(UAS)x4-tk-CAT and wild-type Gal-erbA with a 10-fold excess of expression vectors for the Gal4-DBD or mutant Gal-ErbA fusions was performed into L-tk- cells, as indicated. Fold repression relative to Gal4-DBD alone is shown.

 
To check the functional integrity of the different mutants, we combined the wild-type Gal-ErbA with a 10-fold excess of Gal4-DBD or the Gal-ErbA mutants. Cotransfection of Gal-ErbA with the Gal4-DBD expression vector resulted in a significant reduction of its silencing (from 20- to 4-fold repression) probably by formation of inactive heterodimers, while coexpression of the P398R or L489R mutants hardly affected the silencing function (Fig. 2CGo). Thus, wild-type/mutant Gal-erbA heterodimers appear to be functional, suggesting that the overall structure of the mutant silencing domains is not destroyed.

Mutation in SSD2 of the V-Erba Silencing Domain Completely Abolishes in Vitro Interaction with the SMRT Corepressor
To understand the differential behavior of defective v-ErbA mutants in the complementation experiments, we decided to investigate directly the interaction between the wild-type or mutant v-ErbA repression domain and corepressors. We used a bacterially expressed fusion protein between glutathione-S-transferase (GST) and the C-terminal receptor interaction domain of SMRT [amino acids (aa) 980-1495] which was affinity purified (GST-C.SMRT). The wild type and mutant (see Fig. 1Go) Gal-ErbA fusion proteins were expressed in COS-1 cells, whole cell extracts were prepared and used in electrophoretic mobility shift assay (EMSA) experiments (Fig. 3Go).

Incubation of extracts containing Gal-ErbA fusion proteins in the presence of a Gal4 binding site (UAS) probe resulted in the formation of a specific complex, which was not obtained with extracts from mock-transfected cells (Fig. 3Go, lane 20). Addition of GST-C.SMRT to the wild-type Gal-ErbA led to the appearance of a slower migrating SMRT-ErbA complex (Fig. 3Go, lane 16), supershifting the original DNA-protein complex (lane 15) and thus indicating an interaction between these two factors. No such complex was obtained in the negative controls, testing either the Gal4-DBD alone with GST-C.SMRT (lane 19) or wild-type Gal-ErbA with only GST (lane 17). Point mutants P475R, I537R, and L540R, displaying wild-type silencing activity, are not affected in their interaction with GST-C.SMRT (Fig. 3Go, lanes 6, 10, and 12). Interestingly, the point mutants P396R and P398R within SSD1 and L544R within SSD3 showed a weaker supershift with GST-C.SMRT (lanes 2, 4, and 14) as compared with the one obtained with the wild-type Gal-ErbA, whereas the completely inactive point mutant L489R, located in SSD2, was unable to form a SMRT-receptor complex (Fig. 3Go, lane 8).

In conclusion, in vitro interaction studies of v-ErbA mutants with the corepressor SMRT reveal a correlation between repression function and corepressor interaction.

Correlation between the Silencing Activity and Corepressor Binding of Several Point Mutants of V-ErbA in Vivo
To extend the results concerning the in vitro interaction between v-ErbA mutants and corepressors, we performed in vivo mammalian two-hybrid experiments. We coexpressed the Gal-ErbA silencing domain fusion proteins together with the receptor interaction domain of N-CoR (aa 1585–2453) or SMRT (aa 1073–1495) as VP16 fusions in CV-1 cells. Using the reporter plasmid p(UAS)x5-SV40-LUC, a specific interaction between the Gal-ErbA fusions and the VP16-corepressor protein (VP16-N-CoR-C' or VP16-C.SMRT) results in a high luciferase expression via the transactivation domain of VP16.

Expression of the VP16-corepressor fusion proteins only marginally affected the transcription activation by Gal4-DBD (not shown). We tested three defective point mutants, P398R, L489R, and L544R (Fig. 1Go), each localized in one of the silencing subdomains SSD1–3. The point mutants P398R in SSD1 and L544R in SSD3 exhibit a reduced silencing activity, while mutant P489R within SSD2 was completely inactive (see Fig. 2Go). The wild-type Gal-ErbA displayed a strongly enhanced transcription in the presence of both VP16-N-CoR-C' or VP16-C.SMRT, the interaction with N-CoR being significantly stronger (Fig. 4Go). This observation is consistent with the observed weaker interaction of TR with SMRT as compared with N-CoR (51). The two weakly active mutants, P398R and L544R, showed a reduced interaction with both corepressors, and the extent of the decrease was similar for both N-CoR and SMRT. In contrast, the repression-defective mutant L489R within SSD2 was unable to recruit either of the two corepressors.



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Figure 4. In Vivo Interaction between V-ErbA Mutants and N-CoR or SMRT Depends on SSD2

Mammalian two-hybrid experiments of v-ErbA mutants with the two corepressors N-CoR and SMRT were performed. The indicated wild-type or mutant Gal-ErbA fusions were transiently transfected into CV-1 cells together with the VP16-fusions of the C-terminal receptor interaction domains of N-CoR (aa 1585–2453) or SMRT (aa 1073–1495) and the reporter plasmid p(UAS)x5-SV40-LUC. Protein interactions between the v-ErbA mutants and the corepressors will result in high luciferase activity via the transactivation domain of VP16. The resulting data are presented as fold activation compared with the corresponding Gal-ErbA fusion coexpressed with the transactivation domain VP16 alone. Coexpression of the VP16 activation domain has no effect on the repression levels of the Gal-ErbA fusions (not shown).

 
In conclusion, we can separate the v-ErbA point mutants into different classes according to their ability to interact with the corepressors N-CoR and SMRT: mutants with wild-type activity show wild-type interaction, mutants with a residual silencing activity exhibit a reduced corepressor recruitment, and the completely inactive mutant is unable to interact with any of the corepressors.

Silencing Subdomains Are Not Able to Fully Recruit the Corepressor N-CoR
To analyze the role of the isolated v-ErbA silencing subdomains in corepressor interaction, we first performed GST pull-down experiments. Fusion proteins of the full-length silencing domain of v-ErbA or the three subdomains with the GST were bacterially expressed, coupled to glutathione-Sepharose beads, and used to precipitate in vitro translated 35S-labeled nuclear receptor interaction domain of N-CoR (N-CoR-C', aa 1585–2453). Addition of 35S-labeled luciferase as an internal control showed no nonspecific interaction with any silencing subdomain (not shown). The three subdomain fusions, GST-SSD1 (aa 360–434), GST-SSD2 (aa 434–508), and GST-SSD3 (aa 508–639), exhibit a very weak interaction (Fig. 5Go) compared with that observed with the wild-type full-length silencing domain of v-ErbA (aa 346–639). Interestingly, GST-SSD2 as well as GST-SSD1/2 (aa 362–508), containing both SSD1 and SSD2, showed a slightly higher N-CoR interaction in this very sensitive protein-protein interaction assay. No interaction was observed with GST alone (Fig. 5Go). These results indicate that the isolated subdomains interact marginally with the corepressors in vitro and that SSD2 is able to interact weakly with N-CoR.



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Figure 5. The Isolated Subdomains SSD1–3 Only Weakly Interact with N-CoR in Vitro

GST-pull down experiments between N-CoR and the three silencing subdomains (SSD1–3) of v-ErbA were performed. Bacterially expressed GST or GST fusions with the full-length v-ErbA repression domain or the silencing subdomains SSD1, SSD2, SSD3, and SSD1/2 were tested for interaction with in vitro translated nuclear receptor interaction domain of N-CoR, as indicated. The input lane contains 10% of the 35S-radiolabeled protein used for the interaction studies.

 
Cooperative Binding of the Corepressors N-Cor and SMRT by V-ErbA Silencing Subdomains in Vivo
To analyze the binding of corepressors to the v-ErbA silencing subdomains in vivo, we used the mammalian two-hybrid assay in CV-1 cells. Each of the three silencing subdomains was expressed as a fusion protein with the Gal4-DBD together with VP16-C.SMRT or VP16-N-CoR-C'. The transcriptional activities and the interactions were analyzed by cotransfection with the reporter plasmid p(UAS)x5-SV40-LUC. As expected, none of the subdomains SSD1–3 alone was able to substantially repress transcription, and all three failed to interact with the corepressors N-CoR and SMRT (Fig. 6Go). Combination of two of the three silencing subdomains was not sufficient to restore corepressor binding, consistent with the lack of silencing activity. In contrast, cotransfection of all three silencing subdomain fusions (Gal-SSD1, -SSD2, and -SSD3) resulted in a nearly 5-fold repression and restored the recruitment of both VP16-N-CoR-C' and VP16-C.SMRT. Additionally, in squelching experiments none of the silencing subdomains was able to titrate out corepressors for silencing activity via the full-length v-ErbA repression domain (not shown).



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Figure 6. Silencing Subdomains Bind Corepressors Cooperatively in Vivo

Interaction between the three silencing subdomains (SSD1–3) and the two corepressors, N-CoR and SMRT, was tested in mammalian two-hybrid assays, performed as described in Fig. 4Go. The Gal fusions of the v-ErbA full-length repression domain or the silencing subdomains were cotransfected with the nuclear receptor interaction domains of N-CoR (aa 1585–2453) or SMRT (aa 1073–1495) fused to VP16 and the reporter plasmid p(UAS)x5-tk-LUC into CV-1 cells. For the combinations of the silencing subdomains SSD1–3, expression vectors were cotransfected in equal amounts (0.1 pmol). Amounts of expression vector were kept constant by addition of Gal4-DBD expression vector. Fold repression is shown relative to Gal4-DBD and is measured in the presence of pCMX-VP16. Fold induction is defined as the luciferase activity in the presence of the VP16-corepressor fusion relative to that obtained with VP16 alone. The silencing function of v-ErbA or its subdomains was not affected by expression of VP16, and the VP16-corepressor fusions only marginally affected the basal level of Gal4-DBD (not shown).

 
Our results clearly show that the combination of the three silencing subdomains, which each interact marginally with corepressors, restores the in vivo interaction between the heteromeric transcription factor and the corepressors N-CoR or SMRT, probably involving a cooperative recruitment by the combination of SSD1, SSD2, and SSD3.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We previously showed that the silencing subdomain of v-ErbA consists of three inactive subdomains that restore activity when brought together on the same promoter. We also described different mutations located in each of these subdomains leading to the loss of the silencing function, and we showed that these inactive mutants are unable to compete for silencing cofactors in squelching experiments (50). In a first attempt to understand the function of each of the subdomains in repression, we tested whether an inactive mutant in one of the subdomains could be rescued by coexpression with a mutant in a different subdomain (Fig. 2BGo). Mutant P398R in SSD1 could indeed be complemented by coexpression of mutant L544R in SSD3, while the silencing function of mutant L489R in SSD2 could not be restored by coexpression of any of the other mutants. Coexpression in excess of this mutant does not lead to a dominant impairment of the silencing function of the wild-type Gal-ErbA, in contrast to the Gal4-DBD alone (Fig. 2CGo). This observation, together with the fact that homodimers are readily formed in gel retardation experiments and that their migration is similar to that of the wild-type Gal-ErbA or other mutants (Ref. 50 and Fig. 3Go), strongly suggests that the overall structure of mutant L489R is intact.

We then tested several v-ErbA mutants for their ability to interact with the corepressors N-CoR and SMRT in vitro and in vivo. Supershift experiments and mammalian two-hybrid studies clearly revealed that mutant L489R in SSD2 is unable to recruit corepressors, while mutant P398R in SSD1 or mutant L544R in SSD3 shows a reduced interaction with N-CoR and SMRT (Figs. 3Go and 4Go). These results correlate with the silencing function of the different mutants; mutations in SSD2 completely abolish repression, while mutations in SSD1 or SSD3 only weaken this function. We suggest that the residual repression activity of mutant P398R and L544R, involving their weak interaction with corepressors, is sufficient to restore wild-type interaction and silencing when they are combined. Such an effect would be impossible for the completely inactive mutant L489R in SSD2.

The functional differences between point mutants in SSD1, SSD2, or SSD3 raises the question concerning the precise role of each of these silencing subdomains, which are inactive on their own but synergize in transcriptional repression (50). Previous mutation studies of TR showed that deletion of the hinge region (N-CoR-box in SSD1) (38) or the ninth heptad repeat/helix 11 in SSD3 (47, 48) abolishes its interaction with N-CoR or SMRT. However, these regions were not tested individually for corepressor binding. In GST pull-down experiments we show a weak N-CoR interaction with SSD2 and a very weak N-CoR recruitment by SSD1 or SSD3, as compared with the complete silencing domain of v-ErbA (Fig. 5Go). This result is further supported by the fact that none of the silencing subdomains, when coexpressed as fusion with a nuclear localization signal, was able to titrate corepressors for repression activity via the full-length repression domain of v-ErbA (not shown).

Furthermore, no interaction between the isolated subdomains and N-CoR or SMRT was observed in mammalian two-hybrid experiments (Fig. 6Go). Even the combination of the two previously defined corepressor interaction regions, the N-CoR-box (38) in SSD1 and the ninth heptad repeat/helix 11 in SSD3 (47, 48), is not sufficient for corepressor binding. We show that N-CoR and SMRT are only recruited when all three silencing subdomains (SSD1–3) are combined, regaining repression activity similar to the full-length repression domain. Our studies suggest a cooperative binding of corepressors by the three silencing subdomains, resulting in the formation of a functional repression complex. Isolated SSD1–3 appear to be able to refold in a native-like structure able to present a correct protein surface for corepressor binding.

Recently, the human protein Alien was described as a new corepressor for TR that is structurally unrelated to N-CoR and SMRT (52). Alien is able to interact constitutively with v-erbA and with TR in the absence of hormone. No Alien interaction was observed with repression-defective TR or v-erbA mutants or with the TR SSD1 or SSD2/3 in a yeast two-hybrid experiment. These observations correlate well with our results concerning v-erbA mutants and subdomains, suggesting that the molecular interactions between TR or v-erbA and the corepressors N-CoR, SMRT, and Alien are similar, with only subtle differences.

Analysis of the silencing-deficient mutants allows a closer inspection of the different subdomains. Weakly active mutants are altered in SSD1 and SSD3, both of which contain regions previously shown to interact with corepressors. Mutations in SSD1 (P396R and P398R) are localized in the so-called hinge region, close to the previously described AHT-mutation in TR (38). SSD3 contains the ninth heptad repeat/helix 11 (44), previously shown to be involved in corepressor binding (47, 48). The mutation described here, L544R, is localized in helix 8 (44), suggesting that this region may contribute to corepressor interaction as well. However, these mutants retain some ability to interact with corepressors, consistent with their residual silencing activity. Our results identified SSD2, consisting of helices 3–6 in the TR{alpha} structure (44, 46), as a new important corepressor targeting domain. Mutations in this subdomain most strongly affect silencing by v-ErbA and preclude rescue by mutants in other subdomains. A slightly stronger interaction of SSD2 with N-CoR, as compared with SSD1 and SSD3, was observed in the GST pull-down experiments, which increase the sensitivity for weak protein interactions. In the crystal structure of the apoform of hRXR{alpha} (46) or the holoform of rTR{alpha} (44), the inactivating point mutation of L489R is located within helices 5/6 (50). Interestingly, this location corresponds to the end of the LBD-signature motif [(F/W)AKxxxxFxxxDQxxLL], which was shown to be involved in corepressor binding by Rev-erbA{alpha} and Rev-erbAß (RVR) (53). However, Rev-erbA{alpha} and RVR interaction with N-CoR was mapped to the N-terminal end of this motif, in helix 3 (54), and requires both interaction domains of N-CoR (RID-I and -II), in contrast to TR (54). In addition, corepressor interaction with RVR does not involve an N-CoR-box in region D. These observations suggest that important differences exist in the way Rev-erbAa/RVR interacts with corepressors compared with v-erbA/TR; whether helix 3 in Rev-erbA{alpha} or helices 5/6 in v-erbA are responsible for this divergent behavior remains to be established. Our results present the first evidence for the involvement of helices 5/6 in TR or v-erbA corepressor interaction. These helices are positioned close to helix 1, identified as the corepressor interaction domain N-CoR-box in TR (38). Whether the precise mechanism is the presentation of a larger interaction surface to the corepressor or a mutual structural stabilization of the different subdomains is at present unclear.

Our results indicate a good correlation between the strength of corepressor recruitment and the extent of the silencing activity. However, a mechanism proposing merely an accumulation of corepressor interaction surfaces would be too simplistic. We clearly show that all three subdomains must be present for functional interaction, suggesting that each subdomain has a vital role to play, which cannot be filled by the others. The complexity of the multiprotein complexes involved in the repression process offers numerous additional possibilities for modulation. Each subdomain might make optimal contacts with a different part of the corepressors. A precise sequence of events might be required for high-affinity interaction. The corepressor interaction might be nonproductive, resulting in an impaired interaction with additional cofactors involved in the repression mechanism, such as the Sin3a/HDAC histone deacetylase complex shown to be recruited by the corepressors N-CoR or SMRT (55, 56, 57). Recent studies concluded that the phosphorylation status via the epidermal growth factor signaling pathway is important for binding of the corepressor SMRT to nuclear receptors (58). SMRT, N-CoR, and Sin3a were shown to interact with TFIIB (59, 60), which was previously also described to bind TR (29). Further investigations will be required to understand the precise arrangement of all these factors and their role in transcriptional repression.

In conclusion, the silencing subdomain SSD2 is a new targeting domain for corepressor interaction of v-erbA, which acts only in the entire repression domain of v-ErbA. In our model we assume that the identified corepressor interaction regions, like the N-CoR-box in SSD1, helix 5/6 in SSD2, and the ninth heptad repeat/helix 11 in SSD3, represent different cooperative interfaces for the previously shown nuclear receptor interaction domains RID-I and -II of N-CoR (38, 42, 51) and SMRT (39, 41, 61).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The expression plasmids pGST-SSD1/2 (aa 362–508) and pGST-SSD3 (aa 508–639) were described previously (49). Expression vectors coding for GST-ErbA wt (aa 346–639), GST-SSD1 (aa 360–439), and GST-SSD2 (aa 434–508) were cloned by inserting the SmaI/HindIII inserts from the corresponding pAB-gal plasmids (8, 50) into pGEX-KG-GST. The reporter plasmids used in this study contain four (UASx4-tk-CAT) or five tandem copies (UASx5-SV40-LUC) of a Gal4 binding site (62) inserted in front of the tk-CAT gene (8) or the SV40-LUC gene (63) (kindly provided by R. Thiesen, Rostock, Germany).

The expression plasmids coding for the Gal4-DBD (aa 1–147), wild-type Gal-ErbA (aa 346–639), and the control vector {delta} gal (C) have been described previously (8). Expression plasmids coding for the Gal-ErbA point mutants P396R, P398R, P475R, L489R, I537R, L540R, and L544R, as well as for the three silencing subdomains SSD1–3, Gal-SSD1 (hTRß aa 173–265), Gal-SSD2 (aa 434–508), and Gal-SSD3 (aa 508–639), were described previously (9, 49, 50). The plasmids pGEX-GST-C.SMRT (39), for bacterial expression, and the eukaryotic expression vector for the VP16 activation domain pCMX-VP16 (64) were kindly provided by R. M. Evans (San Diego, CA). The plasmid pSK+(T7)-N-CoR-C' (aa 1585–2453) used for in vitro transcription/translation was cloned as a BamHI fragment from pBKS-N-CoR (38) (kind gift from M. G. Rosenfeld, San Diego, CA) into pSK+. For the mammalian two-hybrid assays, the HindIII/EcoRI fragment containing the nuclear receptor interaction region C.SMRT (aa 1073–1495) was cloned into pCMX-VP16, whereas the homologous region of N-CoR-C' (aa 1585–2453) was cloned as a BamHI-fragment from pSK+(T7)-N-CoR-C' into pCMX-VP16.

Cell Culture and Transfections
L-tk-, CV-1, and COS-1 cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. DNA transfer into CV-1 cells was performed using the calcium phosphate precipitation method (65). For each 10-cm dish with 1 x 106 cells, we used 1.5 pmol reporter plasmid cotransfected with 0.5 pmol expression plasmids. For the mammalian two-hybrid experiments, equal amounts of Gal-fusion plasmid (0.25 pmol) and VP16-fusion plasmid (0.25 pmol) were transfected together with 1.5 pmol of reporter plasmid. Cells were harvested after 72 h, and the luciferase activity in the supernatant was determined as described (66). For the complementation experiments, 1 x 106 L-tk- cells were transfected with 1 pmol reporter and 0.25 pmol of each expression plasmid using the diethylaminoethyl-Dextran method, as described previously (50), and seeded on a 6-cm dish containing 7 ml medium and grown 48 h before harvesting. CAT assays were performed 48 h after transfection as described (67). Transfections were done in duplicate and performed in at least three independent experiments. Transfections into COS-1 cells and the preparation of whole-cell extracts from these transfected cells were described previously (49, 68).

EMSA
EMSA experiments were performed using 20,000 cpm of polynucleotide kinase-labeled UAS-DNA probe, 5 µg whole-cell extract in an incubation mix containing 1 µg of polydeoxyinosinic-deoxycytidylic [p(dIdC)], 6 mM HEPES (pH 7.8), 133 mM KCl, 6% glycerol, 0.6 mM dithiothreitol. The DNA-protein complexes formed were analyzed on a 5% polyacryamide gel in 25 mM Tris, 192 mM Glycin. For the supershift experiments, GST-C.SMRT was expressed in Escherichia coli BL21 cells (69). Protein extracts were prepared as described (49), bound to glutathione-Sepharose 4B beads (Pharmacia Biotech , Piscataway, NJ) in 50 mM Tris, pH 8.0, and eluted in the presence of 10 mM reduced glutathione. GST-C.SMRT protein extracts (600 ng) were incubated 20 min on ice with Gal-receptor fusion proteins before the 32P-labeled UAS-DNA probe was added.

Protein-Protein Interactions in Vitro
Protein-protein interactions were assayed essentially as described previously (49). GST-fusion proteins were expressed in E. coli BL21 cells. Cells were harvested by centrifugation and resuspended in NENT buffer [100 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8.0), 0.5% NP-40]. Cells were lysed by freeze-thaw cycles, and the cellular debris was removed by centrifugation. Glutathione-Sepharose 4B beads (Pharmacia Biotech) were washed with NENT and 10 µl of beads were incubated with 100 µl lysate containing the GST-fusion protein for 30 min at room temperature. Subsequently, the supernatant was removed and the beads were incubated with 15% milk powder in NENT for 10 min at room temperature. The beads were washed twice with 1 ml NENT and once with 1 ml transcription washing buffer [20 mM HEPES (pH 7.9), 60 mM NaCl, 1 mM dithiothreitol, 6 mM MgCl2, 8% glycerine, 0.1 mM EDTA]. The volumes of the different lysates used were adjusted to obtain similar amounts of the fusion proteins, as analyzed by Coomassie-stained SDS-PAGE. In vitro translated and 35S-radiolabeled proteins were obtained using a TNT-kit (Promega Corp., Madison, WI) following the manufacturer’s instructions. Crude lysate (5 µl) was incubated with the beads in 100 µl transcription washing buffer for 1 h at room temperature. In vitro translated luciferase was used as a nonspecific negative control. Finally, the beads were washed (5 x 1 ml NENT buffer), and the proteins were solubilized in SDS-loading buffer and analyzed on SDS-PAGE. Gels were treated with fluorigraphic amplifier reagent (Amersham Pharmacia Biotech, Arlington Heights, IL) and the bands were visualized by autoradiography.


    ACKNOWLEDGMENTS
 
We would like to thank M. G. Rosenfeld for the pBKS-N-CoR plasmid and R.M. Evans for the pGEX-GST-C.SMRT and pCMX-VP16 constructs. We are grateful to K. Krueger for excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Marc Muller, Laboratory of Molecular Biology and Genetic Engineering, Institut de Chimie-B6, University of Liege, B-4000 Sart Tilman, Liege, Belgium.

This work was supported by grants from the Région Wallone (ULg 1815); the Services Fédéraux des Affaires Scientifiques, Techniques et Culturelles (PAI P3–042, P3–044, and P4/30) and Actions de Recherche Concertees (95/00–193); and the Fonds National de la Recherche Scientifique (FNRS) (-3.4537.93 and -9.4569.95). M. M. is a Chercheur qualifié at the FNRS. This work contains part of the Ph.D. thesis of K. Busch.

1 Present adress: Humanbiologie, Universität Frankfurt, Siesmayerstraße 70, D-60323 Frankfurt, Germany. Back

Received for publication April 9, 1999. Revision received September 21, 1999. Accepted for publication October 21, 1999.


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