At Least Three Subdomains of v-erbA Are Involved in Its Silencing Function

Kerstin Busch, Bernd Martin, Aria Baniahmad, Rainer Renkawitz and Marc Muller

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several members of the thyroid hormone receptor (TR) family are able to switch from a transcriptional repressor to a transcriptional activator upon binding of their ligand. The oncogene v-erbA is a variant form of the TR unable to bind hormone and thus acts as a constitutive repressor. We demonstrate, using fusion proteins between the DNA-binding domain of the yeast factor GAL4 and the silencing domains of v-erbA and TRß, that point mutations in three different regions severely affect their repression function. Furthermore, the three regions, each as an inactive fusion protein with the GAL4 DNA-binding domain, restore silencing activity when assembled on the same promoter. These observations define at least three silencing subdomains, SSD1–SSD3, which are involved in the silencing function of v-erbA. We propose a model in which full silencing activity is brought about by the combined interaction of each silencing subdomain with corepressors and/or basal transcription factors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormone receptors (TRs) belong to a large family of regulatory proteins that include receptors for steroid hormones, vitamin D, and vitamin A derivatives. These receptors function as ligand-activated transcription factors that bind to their cognate DNA sequences located in the vicinity of specific genes (1). In general, TR activates transcription in the presence of hormone (T3), but represses transcription of a target gene in the absence of ligand (2). This repression represents a true silencing activity (3), as it is independent of the target promoter and even functions on a minimal promoter containing only a TATA-box (4). Furthermore, a separable silencing domain of the TR could be defined that is active when fused to the heterologous DNA-binding domain (DBD) of the yeast transcription factor GAL4 (5). A similar silencing function is found in the oncogene v-erbA, a viral derivative of the chicken TR that is unable to bind hormone and thus functions as a constitutive repressor on the same target genes (2).

The TR (and v-erbA) silencing domain is localized in the C-terminal hormone binding domain (HBD), which also contains activation functions in the presence of T3 (5). Using deletion analysis on GAL4 DBD fusion proteins, the repression function was assigned to a minimal silencing domain encompassing amino acids (aa) 389–639 of v-erbA (5, 6). Cotransfection experiments of different inactive deletion mutants defined two subdomains that restore silencing when combined in a heterodimeric complex (6, 7). These complementary subdomains consist of aa 173–265 and 265–461 or 362–508 and 508–639, respectively, in human (h)TRß or v-erbA. The C-terminal subdomain of TRß was shown to interact in vitro with the basal transcription factor TFIIB only in the absence of T3 (7). Similarly, interaction with the recently described corepressors N-CoR and SMRT is relieved in the presence of hormone (8, 9, 10). On the other hand, repression was obtained in a reconstituted in vitro transcription system using bacterially expressed TR (11, 12, 13). Little is known about the precise structural requirements for the silencing function in TR or v-erbA.

Here we describe mutations in three distinct regions of the silencing domain of v-erbA and TRß that severely affect the repression function. Furthermore, three subdomains, each as an inactive fusion protein with the GAL4 DBD, restore silencing activity when assembled on the same promoter. These observations define at least three silencing subdomains, SSD1–SSD3, which are involved in the silencing function of v-erbA.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two Prolines in the Hinge Region Are Required for Full Silencing Activity
The region (hinge region) between the v-erbA DBD and the domain homologous to the TR hormone binding domain was shown to contain the transactivation domain {tau}2 (aa 207–217) (14) and to interact directly with the corepressors SMRT and N-CoR (8, 9). In a GAL-v-erbA fusion protein, the deletion of aa 389 to 409 resulted in a complete loss of the silencing function (6). A natural mutant of v-erbA was described previously with a change of Pro398 to Arg abolishing the silencing function (15).

We generated the P398R and other point mutations in this region (Fig. 1AGo) in a fusion protein of the GAL4 DBD with the silencing domain of v-erbA and tested their effect on a UAS-tkCAT reporter gene in L-tk- and CV-1 cells. Fold repression was determined relative to the promoter activity obtained after coexpressing only the GAL4 DBD protein.



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Figure 1. Mutations in the Hinge Region Affect Silencing by v-erbA

A, Amino acid sequence alignment of hTRß, rTR{alpha}, hRXR{alpha}, and v-erbA in the hinge region. The positions of {tau}2 in hTRß, helix H1 in rTR{alpha}, and helices H1 and H2 in hRXR{alpha} are indicated, as well as the position of the mutations in v-erbA. B, L-tk- cells (black boxes) or CV-1 cells (stippled boxes) were cotransfected with the reporter pUAS-tkCAT and expression vectors for the fusion protein GAL-erb 346 (WT) or single point mutants thereoff. The graph represents fold repression obtained after coexpression of the wild type or mutant GAL4-v-erbA fusion proteins relative to coexpression of the GAL4 DBD (GAL) alone.

 
Mutation of Pro 398 to Arg resulted in a 3- to 4-fold decrease of the repression function in both cell types (Fig. 1BGo). A similar mutation at Pro 396, very close to the first one (see Fig. 1AGo), leads to a similar decrease in the silencing capacity, suggesting that the overall structure flanking Pro 396 and 398 is required for silencing function in v-erbA and TR. In contrast, changing the hydrophobic Iso 389 to Arg did not affect the repression function of v-erbA (Fig. 1BGo).

Point Mutations in the Ti-Region Abolish Silencing Function
The previously identified Ti-region is highly conserved among the members of the TR family; an internal deletion of this region was shown to severely reduce silencing of v-erbA (5, 6). It covers the structures defined as helices H3, H4, and H5/H6 of rat (r)TR{alpha} (16) (Fig. 2AGo), which present a clear amphipathic character and would therefore be good candidates for a protein-protein interaction interface. Mutations in this region affected the repression function in different ways (Fig. 2BGo). While the change of Pro 475 to Arg had no (in L-tk- cells) or a marginal (in CV-1 cells) effect, mutation of Pro 481 to Arg drastically reduced the silencing ability. The exchange of Leu 489 to Arg similarly abolished repression, whereas mutation of the Cys 493 to Leu resulted in close to wild type activity, in both cell types tested.



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Figure 2. Mutations in the Central Ti Region of v-erbA Abolish Its Repression Function

A, Sequence alignment displaying helices H3, H4, H5, and H6 in rTR{alpha}, the homologous regions in hTRß and hRXR{alpha}, and the position of the mutations in v-erbA. B, Fold repression relative to coexpression of GAL4 DBD (GAL) obtained by coexpression of wild type (WT) or mutant GAL-erb346 as indicated (see also legend to Fig. 1Go).

 
Our results indicate that amino acids corresponding to the interhelical region H4 to H5 and to the N-terminal part of H5 of the TR{alpha} are of great importance for the silencing function of v-erbA.

Helix 8 Is Involved in the Silencing Function of v-erbA
An interesting feature in the C-terminal half of the v-erbA silencing domain is the region corresponding to helices H8 and H9 of rTR{alpha} (16) (Fig. 3AGo), as it corresponds to the activation domain {tau}3 in hTRß (aa 339–368) (14) and is also highly conserved among nuclear receptors. On the other hand, H8 and H9 could be involved in the dimerization function of TR, as was shown for the homologous region in human retinoid X receptor-{alpha} (hRXR{alpha}) (17). Therefore, we concentrated on H8 and particularly on the amphipathic structure formed by helix H8. Leucines and one isoleucine, all located on the same side of the {alpha}-helix, were changed to the basic arginine, and the repression function of the mutant GAL4-fusion proteins was tested (Fig. 3BGo). While mutants L530R, I537R, and L540R displayed wild type activity, replacement of Leu 544 by Arg resulted in a 3-fold decrease of the silencing activity. Similar results were obtained using L-tk- or CV-1 cells. Thus, the C-terminal part of helix H8 contributes to the repression function of v-erbA.



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Figure 3. Silencing Mediated by Mutants in the {tau}3 Region of the v-erbA- Silencing Domain

A, Sequence alignment of the {tau}3 domain in hTRß, rTR{alpha} helices H8 and H9, and the homologous region in hRXR{alpha} and v-erbA. The mutations in the v-erbA silencing domain are indicated. B, Fold repression mediated by the wild type (WT) or mutant GAL-erb 346 relative to the GAL4 DBD (GAL) in L-tk- cells (black boxes) and CV-1 cells (stippled boxes).

 
The Mutant Phenotype Is Seen in the Context of the Full Length v-erbA
The v-erbA mutant P398R was previously described to be completely deficient in silencing activity in cotransfection experiments (15). In our system, the GAL-v-erbA fusion protein mutant P398R still retained some activity (6-fold repression). To understand the reasons for this discrepancy, we decided to test some of the mutants in the context of the full length v-erbA protein using as a reporter gene the tkCAT fusion controlled by three copies of the everted palindromic binding site F2 (4). Expression of the wild type v-erbA results in a 4-fold repression (Fig. 4Go), as did the functional mutant P475R (compare with the 20-fold repression obtained using GAL4 fusions). In this assay, mutant P398R displayed only a marginal activity, while mutants L544R, P481R, and P396R had no effect on transcription. We conclude that the use of GAL4 fusion proteins represents a much more sensitive method to study transcriptional effects, allowing in particular a finer classification of the mutants.



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Figure 4. The Mutant Phenotype Is Conserved in the Full Length v-erbA

CV-1 cells were cotransfected with the reporter plasmid (F2)x3-tkCAT and wild type (WT) or mutant full-length v-erbA expression vectors. The graph represents fold repression relative to cotransfection with an empty expression vector (C).

 
Mutations in the Ti-Region and {tau}3 Affect the Silencing Function of rTRß
To detect possible relationships between the silencing function without ligand and the activation function of TR in the presence of ligand, we decided to analyze rTRß mutants for their repression and activation capacity in our system. Some of the mutants we used were shown to superactivate a reporter gene in the absence of hormone, as compared with wild type, when tested in yeast (18), possibly reflecting a lack of silencing activity in higher eukaryotes. To test this possibility, we constructed fusion genes coding for the GAL4 DBD and the HBD of wild type (WT) and mutant TRß (see Fig. 5AGo). After transfection into L-tk- or CV-1 cells, the ability of the chimeric proteins to repress or activate transcription of a UAS-tkCAT gene was assessed. The wild type GAL-hTRß led to a 7- or 5-fold repression in the absence of ligand, and a 15- or 40-fold activation in the presence of T3, in L-tk- or CV-1 cells, respectively (Fig. 5BGo). All of the mutants displayed close to wild type transactivation function in the presence of hormone, but mutants K419E, K415E, and V279E/K283R/K301Q seem to mediate a slightly reduced ligand-dependent activation in CV-1 cells. Interestingly, the superactivation described in yeast is not detected in higher eukaryotic cells. In contrast, three of the mutants (K419E, K415E, and V279E/K283R/K301Q) displayed a 3- to 4-fold lower silencing activity as compared with wild type. Mutant N359S showed close to wild type repression in CV-1 cells. These results support the involvement of the Ti region and of helix H8 in the silencing function, and they further point to helix H11 (see Fig. 5AGo) as an important component of the silencing domain. On the other hand, the fact that none of the TRß mutants displayed an important difference in its activation capacity clearly shows that both functions, transactivation and silencing, represent two independent activities of the same protein.



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Figure 5. Several Mutations in the TRß HBD Affect Silencing, but Not Transactivation

A, Amino acid sequences of the concerned regions (Ti, {tau}3, and helix 11) in TRß. The point mutations in the repression domain of TRß are indicated. L-tk- (black boxes) and CV-1 cells (stippled boxes) are cotransfected with the reporter pUAS-tkCAT and the expression plasmids coding for the GAL4 DBD (GAL), the fusion proteins GAL94-hTRß (aa 173–461) (WT), or the GAL147-rTRß point mutants (aa 172–456). B, The graph represents fold repression of the WT or the different mutants of GAL-TRß relative to GAL. C, Fold activation mediated by the WT or the mutants of GAL-TRß relative to the GAL4 DBD (GAL).

 
Expression and DNA Binding of the Fusion Proteins
To ensure that the lack of activity of the defective mutants was not due to a lower expression or a defect in DNA binding of the mutant, we expressed the fusion proteins in COS-1 cells and prepared whole cell extracts of the transfected cells. Electrophoretic mobility shift assays were performed using an oligonucleotide containing a GAL4 binding site as a probe. Each of the extracts gave rise to a specific DNA-protein complex (Fig. 6Go) not present in extracts from mock-transfected cells (lane 19). The complexes can be competed by the unlabeled specific oligonucleotide, but not with an unrelated sequence (data not shown). A faster migrating complex was formed using each extract, presumably originating from a degraded GAL4 DBD (lane 13), as it was specific and similar for every extract. Expression of GAL-v-erbA (lanes 1–12) or GAL-TRß (lanes 14–18) fusion proteins gave rise to complexes of lower mobility, as expected. The results show that expression and DNA binding are similar for the wild type and each of the mutants.



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Figure 6. Expression and DNA Binding of the Mutants of v-erbA and TRß Fusion Proteins

Gel retardation analysis using whole cell extracts from transfected COS-1 cells on a polynucleotide kinase-labeled UAS DNA probe (GAL4-binding site). COS-1 cells are transfected with the expression plasmids of GAL4 DBD (aa 1–147) (lane 13) or the different fusion proteins, GAL-erb 346 wild type (lane 12), point mutants thereof (lane 1–11), the GAL-hTRß wild type (lane 18), and the GAL-rTRß-mutants (lanes 14–17). Note that the GAL-hTRß wild type fusion consists of the GAL4 DBD (aa 1–94) and the repression domain of hTRß (aa 173–461). Lane 19 shows the whole cell extract of COS-1 cells transfected with an empty expression vector (C).

 
Three Subdomains Cooperate for Silencing Function when Placed on Different Molecules
Our mutational analysis defined three regions that seemed to be important for silencing function. Therefore we decided to test whether the silencing domain of v-erbA could be split into three regions (Fig. 7AGo), which would restore repression when combined. We showed previously that different GAL-v-erbA chimeras are able to heterodimerize via the GAL4 region aa 1–147 (6). These heterodimers, appropriately matched, were able to mediate silencing on a reporter plasmid (6) controlled by a single palindromic GAL4-binding site (UAS). To assemble simultaneously three different fusion proteins on a regulatory region, we decided to use the tkCAT transcription unit placed downstream of a hexamerized UAS. This construct can bind up to six fusion protein dimers; thus each of the three tested chimeras should be present.



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Figure 7. Three Subdomains Cooperate for Silencing Function When Placed on Different Molecules

A, Diagram of the linear structure of hTRß, v-erbA, and the GAL4 DBD (GAL) fusions with the different C-terminal subdomains of hTRß and v-erbA. Note that SSD1 was derived from hTRß, as it is functional in this type of experiment, whereas the other silencing subdomains (SSD 1/2, 2/3, 2 and 3) are parts of the repression domain of v-erbA. B, L-tk- cells are cotransfected with the reporter plasmid (UAS)x6-tkCAT and several combinations of the indicated C-terminal subdomains of hTRß and v-erbA fused to the GAL4 DBD. Fold repression of the GAL-erb 346 wild type (WT) and the different combinations is relative to GAL.

 
The experiments were carried out in L-tk- cells by cotransfecting the reporter plasmid (UAS)x6-tkCAT together with three expression vectors coding for different GAL-v-erbA or GAL-TRß fusion proteins. We decided to use the TRß fusion protein for GAL-SSD1 (aa 173–265), as this domain was shown before to form a silencing subdomain (7), whereas the other silencing subdomains are taken from v-erbA. In the controls, when no, one, or two chimeras were expressed, the total amount of transfected DNA was kept constant. The results are presented in Fig. 7BGo. The repression is indicated relative to transfection of pGAL4 DBD alone. Transfection of 0.6 pmol of pGAL-v-erbA, coding for a fusion protein containing the complete v-erbA-silencing domain, leads to 18-fold repression of the basal level, as expected. Expression of each of the chimeric proteins, GAL-SSD1, GAL-SSD2, GAL-SSD3, GAL-SSD1/2, or GAL-SSD2/3 alone, does not result in any silencing effect. Coexpression of GAL-SSD1 + GAL-SSD2/3 or GAL-SSD1/2 + GAL-SSD3 resulted in a synergism between the expressed subdomains leading to 11-fold or 14-fold repression, respectively. Synergism in this system is strong enough to yield silencing effects similar to the one obtained with the complete silencing domain. Neither of the other dual combinations led to a significant repression. In contrast, coexpression of the three fusion proteins, GAL-SSD1, GAL-SSD2, and GAL-SSD3, results in 6-fold silencing.

These results suggest that the simultaneous presence of the three v-erbA subdomains, SSD1, SSD2, and SSD3, restores silencing activity, whereas each one alone or any combination of two is nonfunctional.

The silencing domain of v-erbA is thus composed of three defined subdomains, all of which represent separable structural entities and which cooperate to result in the repression function.

Competition for Silencing Cofactors Requires the Intact, Full-Length Silencing Domain of v-erbA
To test for the possible involvement of (a) corepressor(s) in silencing activity, we performed cotransfection experiments in which we expressed GAL-erb 346 as a silencer protein and large amounts of the silencing domain of v-erbA (WT) or functionally characterized point mutants as competitors in L-tk--cells. Relief of silencing was tested on the p(UAS)-tkCAT reporter plasmid (Fig. 8Go).



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Figure 8. Relief of Silencing Activity by in Vivo Competition Experiments

Cotransfection experiments in L-tk- cells with the reporter plasmid UAS-tkCAT, the expression plasmid pGAL-erb 346, and wild type (WT) or mutant ligand binding domain of v-erbA as competitor. Repression activity is mediated by cotransfection of pGAL-erb 346 (280 ng) with the empty expression plasmid (C) (10 µg) in comparison to GAL. Silencing activity obtained after additional cotransfection of an excess (10 µg) of expression vector coding for the v-erbA-silencing domains is shown for wild type (WT) and each mutant.

 
Transfection of pGAL-erb 346 (280 ng) together with 10 µg empty pAB-{Delta}gal (C) led to an 11-fold repression of the CAT activity as compared with transfection of pGAL4-DBD. This repression was relieved more than 4-fold by coexpression of the wild type v-erbA silencing domain (WT), showing the requirement of one or several titratable corepressors for silencing activity. In contrast, mutants P396R and P398R, located in SSD1, were clearly unable to titrate out the cofactor(s), in correlation with their weak silencing activity (see Fig. 1Go). The next point mutation cluster is located in the Ti-region (Fig. 2Go). The active mutant P475R clearly displays an ability to relief silencing, while the mutants P481R and L489R, which show no silencing activity (Fig. 2Go), do not compete for (a) corepressor(s). Point mutants L530R and I537R, located within the {tau}3-region and presenting wild type repression function, also compete the silencing activity. Mutant L540R still shows silencing activity and displays a weak competition function, but the nearly inactive mutant L544R does not relieve the repression activity of v-erbA.

In conclusion, our results show that the full length silencing domain of v-erbA is able to relieve the silencing activity of GAL-erb 346. Mutants presenting a wild type repression function are also able to titrate out (a) corepressor(s), although at different levels. Most importantly, none of the inactive mutants is able to compete for (a) silencing corepressor(s), again supporting the view that the complete, intact silencing domain is required for repression function.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our study suggests that the silencing domain of v-erbA is composed of at least three functional subdomains, each of which is inactive on its own, but which synergize in transcriptional repression. Two lines of evidence support this conclusion. First, we describe inactivating point mutations in each of the proposed subdomains. Second, the combination of the three subdomains, on different molecules bound to the same promoter, restores the silencing function.

In our experiments, we used fusion proteins consisting of the C-terminal v-erbA silencing domain joined to the DBD of the yeast GAL4 transcription factor. The GAL4 DBD ensures correct nuclear translocation (19) and DNA binding to the specific UAS sequence (20). In addition, we previously showed that homo- and heterodimerization of these fusion proteins is mediated by the dimerization activity of GAL4 (6). Moreover, we clearly show, using gel retardation experiments with transfected cell extracts, that the mutations in the silencing domain do not affect the synthesis or DNA binding of the fusion proteins.

A further advantage in using GAL4 fusion proteins is the increased sensitivity with respect to silencing capacity. Mutants P398R, P396R, and L544R are completely inactive when tested in the full-length protein, but still present a 10- to 15-fold repression ability in the GAL4 fusions. In contrast, mutants P481R and L489R retain only a severely reduced (in L-tk- cells) or no (in CV-1 cells) silencing function as GAL4 fusion proteins, while they are indistinguishable from the other inactive mutants when tested in the natural context. This approach thus allows a more precise evaluation of the effects of individual mutations on the repression function.

Trans-acting complementation analysis of individual protein domains has been successfully performed in several cases (6, 7, 21, 22). Here we use a similar strategy to test three different, nonoverlapping subdomains of v-erbA and TRß. To achieve the simultaneous presence of the three different fusion proteins on the same promoter, we used a reporter plasmid controlled by multimerized UAS sequences. Based on the different heterodimer combinations, each subdomain is expected to be bound to the promoter. Indeed, we could observe a recovery of silencing function only upon coexpression of the three subdomain fusion proteins. Our result suggests that the proposed subdomains are able to adopt a functional conformation when isolated from the rest of the protein. This approach might prove useful in the future study of other multifunctional proteins.

We tested the effects on silencing activity of single amino acid substitutions in three regions of the v-erbA-silencing domain. The most N-terminal one (SSD1, aa 173–265) corresponds to the TR hinge region and transactivation domain {tau}2. This region was recently shown to interact with the corepressors N-CoR (8) and SMRT (9). In a GAL-v-erbA fusion protein, the deletion of aa 389 to 409 resulted in a complete loss of the silencing function (6). A natural mutant of v-erbA was described previously with a change of Pro 398 to Arg abolishing the silencing function (15). A homologous mutation in the TR{alpha} similarly abolished its repression function in the absence of ligand, without affecting its ability to activate transcription in the presence of hormone. The authors proposed Pro 398 to be required for the precise positioning of the structures flanking it. Our data confirm this finding; in addition, we show that Pro 396 is required for silencing function as well, supporting the view that these amino acids form a backbone to precisely arrange the {alpha}-helical structures flanking them. It is unclear whether the prolines are directly involved in protein-protein interactions in addition to this putative structural role, but it was shown that a mutation corresponding to the P398R does abolish the TR interaction with the corepressor SMRT (9). In addition, mutation of amino acids AHxxT at the end of helix 1 in hTRß (see Fig. 1Go) abolished both repression and interaction with N-CoR (8). Surprisingly, mutation of only HxxT at the same site to AxxA results in a silencing domain able to compete for a corepressor (23). The same authors describe a mutant V174A/D177A (corresponding to positions 363 and 365 in v-erbA) that is unable both to repress transcription and to interact with the corepressor. We show that isoleucine 389, located in the same region, is not involved in silencing.

The second domain is identified by the inactive v-erbA mutants P481R and L489R and the triple TRß mutant V279E/K283R/K301Q. It covers the highly conserved, so-called Ti region-spanning helices H4, H5, and H6 in TR{alpha} (16). These mutants appear to be most strongly affected in their silencing activity, pointing to a crucial role of this region in silencing function. Helices H5 and H6 form a highly hydrophobic surface (see the arrangement of L and I in Fig. 2AGo) which would be disrupted by the mutations. Insertion of an additional L in mutant C493L has no effect on repression function.

The {tau}3 region was previously defined as a transactivation domain in hTRß (14) and was shown to be involved in T3 binding and heterodimerization with RXR (24). A mutation in this region of v-erbA (mutant L544R) clearly affected the silencing function as well, again altering the amphipathic character of an {alpha}-helix (H8).

An other functional region is suggested by the rTRß mutants K415E and K419E located in helix H11. This region corresponds to the previously described ninth heptad repeat and was shown to be involved in homodimerization and heterodimerization with RXR (25). Furthermore, mutation L365R of the cTR{alpha} (position 605 in v-erbA) results in loss of repression function (25, 26). As a slight effect of these mutations is also observed on induction in the presence of T3, at least in CV-1 cells, we cannot rule out the possibility that a lack of interaction between the silencing domains in the homodimers of the GAL-TRß chimeric proteins is the basis for their reduced activities. These and other mutants have been described to act as superactivators in yeast (18). Our results, showing a similar hormone induction of the mutants compared with wild type TRß in mammalian cells, are consistent with those obtained by Uppaluri et al., 1995 (18). The authors propose that their selection for highly activating receptors in yeast resulted in the identification of TRs adapted to the yeast transcriptional machinery. As a result of this adaptation, these receptors are less well suited for activation in mammalian cells. Here we show that these mutants, in addition, lose their silencing function. Recently, it was shown that coexpression of hormone-binding deficient TR mutants or of v-erbA is able to enhance the hormone-dependent activation by GAL-TR fusion protein (27) in HeLa cells, suggesting that an inhibitory factor interacts with the TR even in the presence of T3. Similarly, a mutation abolishing the interaction of the TR with the putative inhibitor would result in a superactivation. Such a superactivation in mammalian cells, due to the loss of a residual silencing activity in the presence of T3, is not observed. It is unclear whether the effect could be masked by the concomitant loss of activation capacity of the mutants.

Our cotransfection experiments clearly show that the assembly of the three defined subdomains in heteromeric complexes on the promoter of a reporter gene restores silencing function. In particular, the combination of three GAL4 DBD fusion proteins, each containing a different subdomain, restores repression, suggesting that each subdomain is able to adopt an active conformation on its own.

To visualize a potential protein-protein interaction region in the v-erbA/TR silencing domain, we wanted to localize the mutations abolishing the repression function in the three-dimensional structure of the receptor. The structure has been determined for three nuclear receptors, hRXR{alpha} (17), hRAR-{gamma} (28), and rTR{alpha}1 (16), only one of which (hRXR{alpha}) was generated in the absence of ligand. As the structure of different receptors is quite similar, in contrast to the conformational change induced in each receptor upon ligand binding, we used the structure determined for RXR in the absence of ligand.

Figure 9Go shows the RXR-ligand-binding domain structure with the highlighted (red) positions of the amino acids involved in silencing. Strikingly, these amino acids are all located in one region of the HBD, opposite to the C-terminal activation domain {tau}4/AF-2AD (14, 29, 30). Moreover, they all face to the outside of the molecule, supporting the notion that these regions represent interaction interfaces with other factors. Helices H1 and H2, located in subdomain SSD1, are shown in yellow. Unfortunately, the arrangement of the hinge region is so far unknown, and no information on the structure of the complete SSD1 is available. Subdomains SSD2 and SSD3 are represented in violet and green, respectively. The position of the amino acids required for silencing in the subdomains is consistent with a model where each subdomain, when isolated in a GAL4 DBD fusion protein, is able to adopt a conformation leading to the correct spatial arrangement of the crucial amino acids.



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Figure 9. Location of Several Point Mutants of v-erbA and TRß in the Crystal Structure of the Unliganded Hormone Binding Domain of hRXR{alpha}

Three-dimensional structure of the unliganded hRXR{alpha} HBD. The localization of the functionally analyzed point mutants of TRß and v-erbA are indicated. The positions involved in silencing are indicated in red, while the positions not involved are in light blue. The three identified silencing subdomains SSD1, SSD2, and SSD3 are represented in yellow, violet, and green, respectively. The alignments used to define the positions in the hRXR{alpha} corresponding to the mutated sites in v-erbA or rTRß are represented in Figs. 1Go, 2Go, and 3Go. For the rTRß mutations in helix H11, where amino acids 415–419 in rTRß were aligned to positions 417–421 in hRXR{alpha}.

 
Our results led us to propose a model for the interactions responsible for the silencing mechanism by TR/v-erbA. SSD1 was shown to interact with the corepressors N-CoR and SMRT only in the absence of hormone. On the other hand, it was shown that SSD1 alone, fused to the GAL4 DBD, is unable to repress a UAS-containing reporter gene. The observation that the other subdomains, SSD2 and SSD3, are necessary to restore silencing activity suggests that other interactions are absolutely required for function. In additional experiments, we found that none of the silencing-deficient mutants (Fig. 8Go) and none of the silencing subdomains (data not shown) is able to titrate out (a) corepressing factor(s), further supporting the view that all three subdomains are required for functional corepressor interaction(s). A repression model in which each subdomain interacts with a different corepressor would imply that each mutant deficient in only one subdomain should be able to titrate out the other cofactors, thus resulting in a relief of silencing. This does not appear to be the case, although we cannot completely rule out the possibility that more than one cofactors are involved in silencing. The fact that some of the still active mutants display a reduced capacity to relief silencing indicates, however, that small changes in corepressor interaction might occur without affecting silencing function.

Previously, Hörlein et al. (8) performed interaction studies between TRß deletion mutants and N-CoR, using GST pull down and yeast two-hybrid experiments (8). They showed that deletion of aa 203–230 (part of SSD1) abolishes interaction with N-CoR, while deletion of either aa 260–335 (SSD2) or aa 335–456 (SSD3) clearly weakened the interaction as compared with the full-length ligand-binding domain. These results are consistent with a model where interactions of intermediary factors with one single subdomain are too weak for repression activity, but are stabilized in the presence of the other subdomains to result in a functional silencing complex.

The C-terminal region of TRß, corresponding to SSD2/3, was previously shown to interact with the general transcription factor TFIIB in the absence of ligand (7), and this interaction was shown to be involved in repression by TR in in vitro transcription (11, 12, 13). Thus, a complex picture of transcriptional silencing emerges involving interaction of SSD1 with N-CoR and/or SMRT, strengthening of this interaction by other subdomains, and interaction of SSD2 and/or SSD3 with TFIIB or with a still unknown factor. Further experiments will be required to ultimately understand the precise molecular interactions leading to transcriptional silencing.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
The reporter plasmids used in this study contain a single (UAS-tkCAT) or six tandem copies (UASx6-tkCAT) of a GAL4 binding site (20) inserted in front of the tkCAT gene (5). The reporter F2x3-tkCAT was described previously (4).

The expression plasmids coding for the GAL4 DBD (aa 1–147), GAL-erb 346, GAL-erb 362–508 (SSD1/2), GAL-erb 508–639 (SSD3), and GAL-erb 434 (SSD2/3), as well as the control vector {Delta}gal (C), have been previously described (4). The expression plasmid pGAL-erb 434–508 (SSD2) was obtained by cutting pGAL-erb 362–508 with PvuII and EcoRV and religating, thereby destroying these restriction sites. The pABGAL94, pABGAL94-hTRß (aa 173–461), and pABGAL94-hTRß 173–265 (SSD1) vectors were described previously (14).

For the competition experiments, the PvuII/BamHI fragments from wild type and mutant pABGAL147-erb 346 were cloned into the expression vector pAB-{Delta}gal, resulting in expression vectors for wild type and mutant silencing domains.

The rat TRß mutants were kindly provided by H. C. Towle (18): pG2M/GAL-TRß (aa 172–456)-S4 (K419E), -S10 (K415E), -S20 (V279E, K283R, K301Q), and -112 (N359S) are cloned into the pABGAL147 vector by digestion with HpaI/SalI to obtain the corresponding GAL-TRß mutant fusion proteins.

Expression vectors for the full-length v-erbA mutants were constructed by cloning the gag-coding KpnI/PvuII fragment from pRSV-v-erbA (2) in the KpnI/PvuII-digested expression plasmid pGAL-erb 346 mutant, thus replacing the GAL4 DBD with the original v-erbA N-terminus.

Site-Directed Mutagenesis
The v-erbA-mutants were generated by site-directed mutagenesis as described (31). Briefly, a single-stranded uracil-containing template containing sequences coding for v-erbA aa 346–639 was prepared and annealed with a specific primer carrying the mutation. The annealed primer was elongated using T4 DNA-Polymerase (in the presence of the single-strand binding protein gene 32 from phage T4). After ligation, the product was transformed into Escherichia coli.

The resulting point mutants were confirmed by sequencing. The primers used to obtain the 11 mutants are shown below; the modified bases are underlined. I 389 R: 5'-G-GAG-GAG-ATG-AGG-AAA-TCC-CTG-C-3' P 396 R: 5'-G-CAC-CGG-CGC-AGC-CCC-3' P 398 R: 5'-GG-CCC-AGC-CGC-AGC-GCA-GAG-G-3' P 475 R: 5'-GCC-AAA-AAC-CTG-CGC-ATG-TTC-TCG-G-3' P 481 R: 5'-C-TCG-GAG-CTG-CGG-TGC-GAG-GAT-CAG-3' L 489 R: 5'-CAG-ATC-ATC-CTG-CGG-AAG-GGC-TGC-3' C 493 L: 5'-G-AAG-GGC-TGC-TTG-ATG-GAG-ATC-ATG-3' L 530 R: 5'-C-GGA-GGG-CGG-GGG-GTC-G-3' I 537 R: 5'-C-GTG-TCT-GAT-GCC-AGG-TTC-GAC-CTC-G-3' I 540 R: 5'-GCC-ATC-TTC-GAC-CGC-GGC-AAG-TCG-C-3' I 544 R: 5'-C-GGC-AAG-TCG-CGG-TCT-GCC-TTC-AAC-3'

Cell Culture and Transfections
L-tk- cells, CV-1, and COS-1 cells were grown in DMEM (GIBCO, Grand Island, NY) 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 (32). L-tk- cells were transfected using the following protocol (33): 1 x 106 cells were suspended in DNA-diethylaminoethyl-dextran solution (1 pmol reporter and 0.5 pmol expression plasmids) and incubated for 60 min at room temperature. Cells were seeded on a 6-cm dish containing 7 ml medium and grown for 48 h before harvesting. For hormonal induction experiments, the serum was depleted of thyroid hormone by extensive charcoal stripping (34). The cells were kept for at least 24 h in depleted medium before transfection; after transfection 10-6 M T3 was added when indicated. CAT assays were performed as described (35).

Transfections were done in duplicate and performed in at least three independent experiments. Transfections into COS-1 cells were done using a similar diethylaminoethyl-dextran suspension method using 25 µg of DNA on 2 x 106 cells. After 1 h incubation in the DNA solution, a dimethylsulfoxide shock was performed for 3 min, the cells were taken up in 30 ml TBS and 10 ml DMEM, spun down, seeded on a 15-cm dish, and grown for 48 h before harvesting (6).

DNA-Protein-Binding Assays
Whole cell extracts were prepared from COS-1 cells transfected with various expression vectors as described (36). Gel retardation 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 poly-deoxyinosinic-deoxycytidylic acid, 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% polyacrylamide gel in 25 mM Tris, 192 mM Glycin.


    ACKNOWLEDGMENTS
 
We would like to thank K. Krueger for excellent technical assistance. We are grateful to D. Moras for providing the coordinates of the three-dimensional structure of the RXR and to W. Wende, G. Schlauderer, and A. Jeltsch for the computer visualization of the mutants in the RXR structure. Furthermore, we thank H. C. Towle for sending us the TRß mutants.


    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, Belgium.

This work was supported by the Fonds der Chemischen Industrie, the Deutsche Forschungsgemeinschaft (SFB 249), the Services Fédéraux des Affaires Scientifiques, Techniques, et Culturelles PAI P3–042 and PAI P3–044, Fonds National de la Recherche Scientifique (FNRS)-3.4537.93 and 9.4569.95, and the Actions de Recherche concertée-95/00–193. K. Busch was supported by a fellowship from the DAAD (Doktorandenstipendium aus Mitteln des zweiten Hochschulsonder-programmes") and B. Martin by a fellowship from the Boehringer Ingelheim Fonds.

This work contains part of the Ph.D. theses of K. Busch and B. Martin (University of Giessen, Germany).

Received for publication July 26, 1996. Revision received December 20, 1996. Accepted for publication December 30, 1996.


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