Cell-Specific Inhibition of Retinoic Acid Receptor-{alpha} Silencing by the AF2/{tau}c Activation Domain Can Be Overcome by the Corepressor SMRT, But Not by N-CoR

Aria Baniahmad, Uwe Dressel and Rainer Renkawitz

Genetisches Institut der Justus-Liebig Universität D-35392 Giessen, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human retinoic acid receptor {alpha} (hRAR{alpha}) exhibits cell-specific transcriptional activity. Previously, it was shown that in the absence of hormone the wild-type receptor is a transcriptional silencer in L cells, whereas it lacks silencing function and is a weak activator in CV1 cells. Addition of hormone leads to a further increase in transactivation in CV1 cells. Thus, the retinoic acid response mediated by RAR{alpha} is weak in these cells. It was shown that the CV1-specific effect is due to the receptor C terminus. We show, that the failure of silencing by RAR is not due to a general lack of corepressors in CV1 cells, since the silencing domain of RAR is functionally active and exhibits active repression in these cells. Furthermore, we show that the conserved AF2/{tau}c activation function of RAR is responsible for the cell-specific inhibition of silencing. Thereby, the CV1 cell specificity was abolished by replacing AF2/{tau}c of RAR with the corresponding sequence of the thyroid hormone receptor. Thus, we find a new role of the C-terminal conserved activation function AF2/{tau}c in that, specifically, the RAR AF2/{tau}c-sequence is able to prevent silencing of RAR in a cell-specific manner. In addition, we show that the inhibitory effect of AF2/{tau}c in CV1 cells can be overcome by expression of the corepressor SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), but not by that of N-CoR (nuclear receptor corepressor). The expression of these two corepressors, however, had no measurable effect on RAR-mediated silencing in L cells. Thus, the expression of a corepressor can lead to a dramatic increase of hormonal response in a cell-specific manner.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid receptors (RARs) belong to the superfamily of nuclear hormone receptors. They play an important role in various stages of mammalian development and organ differentiation as well as in homeostasis (1). RARs are ligand-regulated transcription factors (for reviews, see Refs. 2–4). In general, RARs are similar to thyroid hormone receptors (TRs) in that they repress gene activity in the absence of hormone (5, 6, 7). Such a repression is mediated by a silencing domain localized in the receptor ligand-binding domain (5, 8). The silencing function (active repression) is, similar to transactivation domains, transferable to other DNA-binding proteins (5) and thus represents a functional domain. It was shown that corepressors are involved in the signaling of silencing from the receptor to the transcription initiation complex (6, 7, 9, 10). In general, hormone binding leads to a conformational change of the receptor, to dissociation of corepressors, to binding of coactivators, and to transcriptional activation.

Nuclear hormone receptors share common structural features. They are composed of a well conserved DNA-binding domain (C region), a variable amino (N) terminus (A/B-region), and a conserved C terminus (D, E, and F region) (for reviews, see Refs. 2, 4, 11, and 12). The receptor C terminus harbors various functions such as silencing, hormone binding, dimerization, and transactivation (4). It was shown that RAR and other nuclear hormone receptors contain at least two activation functions: one is localized in the receptor N terminus and the other in the C terminus. The latter is dependent on the binding of hormone (2, 4, 11, 12). A small stretch of conserved amino acids have been identified to be responsible for ligand-dependent transactivation (3, 4, 9, 13, 14) and is referred to as AF2, AF2-AD, {tau}4, or {tau}c (2, 4, 9, 14, 15). Both activation domains retain their functions when transferred on heterologous DNA-binding proteins (9, 14, 15, 16, 17, 18).

Interestingly, similar to other nuclear hormone receptors, both activation domains of RAR function in a cell-specific manner (19, 20, 21, 22, 23, 24, 25). The detailed mechanisms for these cell-specific hormonal responses are unknown. One explanation may be that receptor-interacting cofactors are distinct from cell to cell. Therefore, the levels or the nature of cofactors may differ, which could lead to cell-specific hormonal responses. Thus, the analysis of cell specificity of retinoic acid responses will shed light into their role in gene regulation and development.

Here, we analyzed the RAR{alpha}, which displays cell-specific transcriptional properties (5, 20, 21, 22). In the absence of hormone, in contrast to the TR, the RAR is a transcriptional activator in CV1 cells. However, RAR acts as a silencer in L cells. Addition of hormone leads to a further activation in CV1 cells, while in L cells transcriptional silencing is relieved and the receptor is converted to a gene activator. In general, the RAR-mediated hormonal response is much weaker in CV1 cells compared with that of L cells. It was shown that the RAR C terminus lacks silencing in CV1 cells and is responsible for the cell type specificity (5).

We show, that the cell specificity is due to the AF2/{tau}c activation domain of RAR and is not due to a general lack of silencing in CV1 cells. Interestingly, silencing in CV1 cells is achieved by replacing RAR-AF2/{tau}c with TR-AF2/{tau}c. This suggests a cell-specific role of AF2/{tau}c for regulation of transcriptional silencing in the context of its receptor. Furthermore, we show that coexpression of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), but not N-CoR (nuclear receptor corepressor), in CV1 cells enhances strongly the hormonal response. Therefore, the transactivation by RAR is reduced in the absence of hormone, and silencing is regained. This indicates that SMRT is one of the limiting factors for RAR-mediated silencing in CV1 cells. Thus, the level of certain cellular corepressors is an important factor in regulation of target genes and the hormonal response of the cell.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RAR{alpha} Is a Transcriptional Activator in the Absence of Hormone in CV1 Cells
In a large number of cell types the retinoic acid receptor (RAR), similar to the TR, acts as a transcriptional silencer in the absence of hormone and in presence of ligand as a hormone-dependent transactivator (3, 5, 31, 32, 33, 34, 35). However, RAR fails to silence promoter activity in CV1 cells in the absence of ligand (5). RAR was shown to harbor two transferable activation domains, both of which act in a cell-specific manner (20, 21, 22, 24).

It was previously shown that the CV1-specific lack of RAR-mediated silencing is localized in its C terminus (Fig. 2Go and Ref.5). Here, we have focused on the RAR C terminus and used the established fusion of the C-terminal part of RAR to the DNA-binding domain of Gal4 (Gal-RAR, Fig. 1Go). This expression plasmid was cotransfected together with a reporter containing the Gal4-binding site [upstream activating sequence (UAS) or 17 mer] in front of the tkCAT fusion. Cotransfection showed that the receptor fusion acted as a transcriptional silencer in the absence of ligand and as a retinoic acid-dependent transactivator in L cells (Fig. 2Go). In contrast, the same fusion acted as a weak activator in CV1 cells even in the absence of ligand (Fig. 2Go). Addition of hormone increased the transcriptional activity. This indicates that the cell-specific effect is localized in the receptor D, E, or F region. Thus, in CV1 cells, retinoic acid induces only a marginal hormonal response by RAR{alpha} (~3-fold), quite different from L cells (~200-fold).



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Figure 2. RAR-AF2/{tau}c Sequence Is Responsible for the Inhibition of Silencing by the Receptor in CV1 Cells

Cotransfection experiments in L and CV1 cell lines of the indicated Gal-RAR expression vectors (0.5 pmol) and the reporter p17mer tkCAT (5) (1.5 pmol). Obtained values were normalized to that of the Gal4-DBD control. Activation above or below the promoter activity is shown as fold activation or fold silencing, respectively. Retinoic acid was added at 10-5 M. White bars and black bars, Values obtained with L cells and CV1 cells, respectively.

 


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Figure 1. Schematic Representation of Expression Vectors Used for DNA Transfection Experiments

Gal fusions and RAR chimeras combined with the transactivations domains of TR-AF2/{tau}c (TR-{tau}4; Ref. 9), p65/NFkB (26), or Oct (26) are shown. Numbers indicate the amino acid position in the original protein. The receptor C, D, and F regions as well as the small activation domain AF2/{tau}c are indicated.

 
Previously, it has been shown that a C-terminal deletion of 60 amino acids (aa), deleting the F region and the activation function AF2/{tau}c of RAR{alpha}, referred to as the RAR silencer core or RAR{Delta}C60, is a constitutive silencer (5, 8). As shown in Fig. 2Go, RAR{Delta}C60 mediates constitutive silencing in both L- and CV1 cells. This indicates that CV1 cells do not completely lack factors required for mediating silencing.

AF2/{tau}c of RAR Is the Cause for the Lack of RAR Silencing in CV1 Cells
To test whether the F region or AF2/{tau}c of RAR is inhibiting silencing in CV1 cells, we generated a receptor missing only the F region (RAR{Delta}C43; Fig. 1Go). As seen in Fig. 2Go, this receptor deletion exhibited similar transcriptional activity as the origin receptor: lack of silencing in CV1 cells in ligand-free conditions and weak hormone induction. Therefore, we conclude that the receptor F region is not required, and that the conserved 17 amino acids (aa 403–419) of RAR AF2/{tau}c are sufficient to inhibit silencing in CV1 cells.

Furthermore, we fused the corresponding AF2/{tau}c of the thyroid hormone receptor (TR-{tau}4 (Ref.9); TR-AF2/{tau}c) to the C terminus of RAR{Delta}C60, creating Gal-RAR-TR-AF2/{tau}c. This receptor chimera acted as a retinoic acid-dependent transcriptional activator in both L- and CV1 cells (Fig. 2Go). Figure 2Go shows that AF2/{tau}c of TR can substitute the activation function of RAR and promote hormone-dependent activation. Interestingly, in the absence of hormone, RAR-TR-AF2/{tau}c acts as a transcriptional silencer in both cell types (Fig. 2Go). Therefore, hormone induction in CV1 cells is enhanced dramatically to about 150-fold, compared with an about 4-fold induction of the original receptor (Fig. 2Go). This suggests that the CV1-specific effect (lack of silencing) is conferred specifically by the RAR AF2/{tau}c domain and not by the homologous domain of TR.

To determine whether the endogenous/intrinsic transactivation function of AF2/{tau}c is responsible for the difference in retinoic acid response between RAR{Delta}C43 and RAR-TR-AF2/{tau}c, we tested for their ability to transactivate in both cell types. As seen in Fig. 3Go, both Gal-RAR402–462 (harboring both AF2/{tau}c and the F region) and Gal-RAR402–419 (RAR-AF2/{tau}c) transactivate only very weakly, if at all, when tested on one UAS element of the reporter 17 mer tkCAT in both cell types. As also seen in Fig. 3Go, AF2/{tau}c derived from TR (TR-AF2/{tau}c) harbors even a stronger transactivation function, at least in CV1 cells, compared with RAR-AF2/{tau}c. Nevertheless, the receptor fusion RAR-TR-AF2/{tau}c exhibits silencing function in CV1 cells. This indicates that the almost constitutive transactivation of RAR in CV1 cells is not caused by a cell-specific activity of AF2/{tau}c.



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Figure 3. The Intrinsic Transactivation of Gal4-Activator Fusions in L or CV1 Cells

Cotransfection of indicated Gal4-activator fusions (1 pmol) together with the reporter p17mer tkCAT (1.5 pmol) in both cell lines. Gal-RAR-AF2/{tau}c and Gal-RAR-TR-AF2/{tau}c encode the receptor-derived aa 402–419 and 445–461, respectively. hTRß sequences are according to the nomenclature (38). Values obtained are normalized to that obtained with Gal4-DBD alone.

 
In addition, we replaced TR-AF2/{tau}c by other small activation domains, such as Oct (aa 143–160, Ref.26), a weak transactivator similar to RAR402–462 (Fig. 3Go), or p65/NFkB (aa 520–550, Ref.26), a strong transactivator similar to TR-AF2/{tau}c. Both RAR-Oct and RAR-p65 fusions showed silencing in the absence of hormone (Fig. 2Go). While the RAR-Oct fusion acted as a silencer, albeit to a lesser extent in the presence of hormone, addition of ligand relieved silencing of the RAR-p65 fusion completely. Although p65 harbors an equally potent transactivation function as TR-AF2/{tau}c in CV1 cells, we did not observe a hormone-dependent activation of the RAR-p65 fusion. This suggests that AF2/{tau}c or a heterologous activation function can be overridden by silencing in the absence of ligand, and thus the degree of intrinsic activation function itself of AF2/{tau}c is not the basis for the CV1-specific effect, in that RAR fails to silence in the absence of hormone.

Taken together, the lack of silencing in the absence of hormone in CV1 cells is due to the specific sequence of RAR-AF2/{tau}c. Our data suggest that the CV1-specific effect is uncoupled from the strength of AF2/{tau}c intrinsic transactivation function, since replacing the conserved sequence AF2/{tau}c of RAR with that of TR rendered the receptor to a transcriptional silencer. Thus, the small AF2/{tau}c sequence of RAR harbors other requirements to inhibit silencing in CV1 cells and exhibit cell type specificity in the context of its own receptor.

The Lack of Silencing by RAR in CV1 Cells Can Be Overcome by the Corepressor SMRT, But Not by N-CoR
Another possibility for the failure of RAR to silence in CV1 cells in the absence of hormone is that one or few corepressors are present in a limiting amount in CV1 cells. These corepressors may be required to overcome the effect of AF2/{tau}c of RAR{alpha}. To test this possibility, we cotransfected the known corepressors SMRT or N-CoR in CV1 cells and tested for hormonal response. In the absence of cotransfected corepressors, RAR exhibited about a 2- to 4-fold ligand-mediated induction (Figs. 2Go and 4AGo). Coexpression of SMRT resulted in a profound hormonal induction of about 22-fold. This was achieved by reducing the activation function by Gal-RAR in the absence of hormone (Fig. 4AGo). As a control, SMRT expression affected the promoter activity only weakly, while it changed the RAR-specific transcriptional activity to a level slightly below that of the promoter activity (Fig. 4AGo). Interestingly, coexpression of N-CoR did not enhance hormonal induction; rather the promoter activity was enhanced in the presence or absence of RAR. Similar results were obtained using Gal-RAR{Delta}C43 in CV1 cells. RAR{Delta}C43 with only the F region deleted lacked silencing function (Fig. 2Go). Coexpression of N-CoR did not affect RAR{Delta}C43 transcriptional properties significantly. In contrast, SMRT coexpression abolished the RAR{Delta}C43-mediated activation and led to transcriptional silencing of about 6-fold in the absence of hormone, while the induced level was unchanged. Thus, a strong hormonal response of above 60-fold was achieved when SMRT was coexpressed in CV1 cells (Fig. 4AGo).



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Figure 4. Hormone Response Is Strongly Enhanced by Coexpression of SMRT in CV1 Cells

A, Gal-RAR (0.5 pmol) was cotransfected with the reporter 17 mer tkCAT (1.5 pmol) and with the expression vector (10 µg) for SMRT or N-CoR or an empty expression vector (C) into CV1 cells. Values obtained with Gal4 DBD alone together with the control vector (C) were set as 1. Retinoic acid, 10-5 M, was used. B, At 10-9 M retinoic acid concentration, hormonal response is greatly enhanced by coexpression of SMRT. Indicated are the fold hormone induction, which is the ratio of values obtained with and without ligand treatment at the indicated hormone concentrations in CV1 cells. Gray bars and black bars represent the cotransfection of the empty vector (C) and the SMRT expression vector, respectively.

 
To test the ligand response at various retinoic acid concentrations, in CV1 cells we compared hormone induction obtained with and without cotransfection of SMRT. As shown in Fig. 4BGo, at concentrations of 10-9 M retinoic acid, the hormonal induction differed significantly and was higher in the presence of SMRT.

This suggests that SMRT is at least one of the cofactors that are limited in CV1 cells for RAR being a transcriptional silencer in the absence of hormone. In addition, we show that the hormone response is strongly enhanced after coexpression of SMRT, but not N-CoR, which indicates that SMRT and N-CoR have differential effects on nuclear hormone receptors. Furthermore, our results indicate that receptor target genes can be regulated by magnitudes with hormone, dependent on the cell type and on the presence of specific cofactors.

Replacing AF2/{tau}c of RAR by that of TR Enhances SMRT Binding to the RAR-Silencing Domain
Replacing the RAR activation domain AF2/{tau}c by that of TR (RAR-TR-AF2/{tau}c) resulted in a receptor fusion that exhibited silencing function in CV1 cells (Fig. 2Go). One explanation for this effect may be that the interaction of SMRT with RAR is affected by exchanging the activation domain AF2/{tau}c. Therefore, we performed band shift experiments using extracts from retinoic acid-free transfected COS-cells. As seen in Fig. 5AGo, addition of bacterially expressed glutathione-S-transferase (GST)-SMRT (30) to COS extracts that expressed Gal-RAR led to a supershift, which represents the receptor-SMRT complex. Addition of the cognate ligand, retinoic acid, abolished the supershift with SMRT, as expected. Previously, we have shown that neither GST nor GST-SMRT alone can bind to the UAS-probe (31). To test the possibility that AF2/{tau}c affects the receptor-SMRT complex formation, increasing amounts of bacterially expressed and purified GST-SMRT (30) were incubated with Gal-RAR or Gal-RAR-TR-AF2/{tau}c containing extracts and with the UAS probe (5). As seen in Fig. 5BGo, both the Gal-RAR or the Gal-RAR-TR-AF2/{tau}c bands were supershifted when SMRT was added, resulting in a GAL-RAR/SMRT complex. Increasing amounts of added GST-SMRT resulted in an increase of each of the receptor-SMRT complexes. However, addition of the same amount of SMRT enabled more of Gal-RAR-TR-AF2/{tau}c than of Gal-RAR to supershift. This suggests that SMRT interacts more strongly with the Gal-RAR-TR-AF2/{tau}c fusion. This is also in accordance with yeast 2 hybrid results (31). Thus, a greater affinity may be the cause for the RAR-TR-AF2/{tau}c fusion to act as a transcriptional silencer in CV1 cells. For this fusion we can speculate that the corepressor SMRT is not limiting in obtaining transcriptional silencing by RAR-TR-AF2/{tau}c.



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Figure 5. Interaction of SMRT with RAR and RAR-TR-{tau}4/AF2 Fusion in Vitro

EMSA analysis was performed with the 32P-labeled gal4-binding site (UAS/17 mer), extracts from transfected COS1 cells, and bacterially expressed SMRT. A, Hormone-sensitive receptor-SMRT complex. Gal-RAR was incubated with bacterially expressed GST (lanes 2 and 3) or GST-SMRT (lanes 4 and 5) in the absence or presence of retinoic acid. A supershift was only obtained when SMRT and the receptor were present in the absence of ligand (lane 4). B, Extracts from Gal-RAR or RAR-TR-{tau}4/AF2-transfected COS1-cells were incubated with increasing amounts of bacterially expressed GST-SMRT (from 25–500 ng) for each receptor fusion. A supershift containing the RAR/SMRT complex occurs only in the presence of SMRT.

 
Thus, the small AF2/{tau}c sequence is not only required for corepressor release in the presence of hormone, but also has an influence on SMRT-receptor interaction in the absence of ligand.

Cellular SMRT Levels May Account for Cell Type Specificity
To analyze the expression levels of SMRT, we performed Western experiments comparing extracts from equal cell numbers of L- and CV1 cells. Using an anti-SMRT antibody directed against the C terminus of SMRT (Santa Cruz Biotechnology, Santa Cruz, CA) we show that the amount of SMRT is distinct between both cell types (Fig. 6Go). The SMRT antibody recognizes in CV1 cells a slightly slower migrating band compared with that of L cells (Fig. 6Go). This indicates that, in addition to endogenous SMRT levels, there may be also cell type-specific variants of SMRT. Both different levels and perhaps variants of SMRT may explain the cell type-specific lack of RAR- mediated silencing in CV1 cells.



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Figure 6. Cell-Specific Amounts of SMRT Detected by Western Blotting

Equal numbers of L or CV1 cells (4 x 105) were used for Western blot analysis using anti-SMRT antibody directed against the SMRT C terminus. The protein marker sizes and SMRT bands are indicated.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our data suggest that both the type of corepressors and the sequence of the AF2/{tau}c activation domain can be a major cause for cell-specific responsiveness to hormone. The RAR C terminus encompassing the receptor C, D, and F region, exhibited no silencing and weak transactivation in the absence of hormone in CV1 cells. In contrast, under the same conditions, RAR acted as a transcriptional silencer in L cells, HeLa (31), and primary chicken fibroblasts (5). One reason for the CV1-specific effect may be unsufficient amounts of corepressor required specifically for RAR-mediated basal level repression or silencing. In accordance with that we show that coexpression of SMRT, but not N-CoR, augmented the hormonal induction dramatically.

Cell-Specific Silencing in the Absence of Hormone
The RAR silencer core (RAR{Delta}C60, aa 143–403) exhibited strong silencing function in the presence or absence of hormone. Although it was shown that this RAR deletion is able to bind all-trans-retinoic acid with similar affinity as that of the wild-type receptor (8, 37). This confirms data that ligand binding is not sufficient to relieve silencing (9, 38). Interestingly, the RAR deletion RAR{Delta}C43, harboring an additional 17 aa, which correspond to its activation function AF2/{tau}c sequence, does not mediate silencing in CV1 cells.

We tested, therefore, whether the strength of the endogenous/intrinsic activation function may be a major cause for the cell specificity. We compared AF2/{tau}c from TR and RAR as well as heterologous activation domains such as that of p65 and Oct2 (26). When we replaced RAR-AF2/{tau}c with TR-AF2/{tau}c, we saw a hormone-dependent receptor transactivation, with a similar level of activity as Gal-RAR or Gal-RAR{Delta}C43. Nevertheless, the intrinsic transactivation of the TR-AF2/{tau}c activation domain, shown in Fig. 3Go, is stronger as that of RAR-AF2/{tau}c. Replacing AF2/{tau}c sequences with the activation domain p65, which had similar intrinsic transactivation properties compared with TR-AF2/{tau}c in CV1 cells, was only able to relieve silencing only by addition of hormone. Similarily, AF2/{tau}c and the F region of RAR was significantly weaker in transactivation, but in the context of the receptor it enabled strong hormone-induced activation. This suggests that the strength of activation function is not the critical event in ligand-induced activation and indicates that strong coactivator binding to AF2/{tau}c, which may displace corepressors in the absence of hormone, can be excluded.

We show, rather, that the levels or type of cellular corepressor SMRT is the cause of the lack of silencing mediated by RAR in CV1 cells. Coexpression of SMRT (6) was able to overcome the lack of silencing of RAR and RAR{Delta}C43 and rendered the receptor into a transcriptional silencer in the absence of hormone, while the level of ligand-induced transactivation of RAR remained unchanged. Thus, our data suggest a direct involvement of SMRT in the magnitude of hormonal response of a cell.

Interestingly, N-CoR was unable to yield similar effects as seen with SMRT, although both proteins are related to each other and share sequence homologies (36). We have shown previously that both SMRT and N-CoR are able to bind to the DNA-bound Gal-RAR fusions (31). While RAR is able to bind to both corepressors, only coexpression of SMRT enhanced silencing. This may indicate that N-CoR, despite the homologies to SMRT, harbors different functions in mediating silencing. Also a difference between the homologous corepressors SMRT and N-CoR was observed for the orphan receptor RevErb (39). SMRT and N-CoR interacted differentially with this orphan receptor: N-CoR, but not SMRT, potentiated the RevErb repression, when bound to DNA.

Another possibility for the cell-specific silencing of RAR may be due to lower levels or to specific variants of the corepressor SMRT in CV1 cells. Based on our Western experiments we have indeed seen a weaker SMRT band in CV1 cells compared with that of L cells. However, there may exist variants of SMRT in CV1 cells. SMRT variants can be generated by differential splicing events or be caused by posttranslational modifications (e.g. phosphorylation). Variants of a corepressor have been described for the SMRT homologous factor N-CoR/RIP13 (40). In any case, we show that the coexpression of SMRT can overcome the endogenous SMRT function. Thus, different SMRT levels and/or variants may be the basis for cell type specificity of receptor-mediated gene repression.

Multiple Functions of Nuclear Receptor AF2/{tau}c Sequence
Our data show that silencing is inhibited by the AF2/{tau}c (17 aa) of RAR specifically in CV1 cells. Fusion of AF2/{tau}c from TR (9) encompassing 17 aa rendered the RAR silencer core into a hormone-dependent transactivator, indicating that the conserved AF2/{tau}c sequences can be exchanged without loss of ligand-induced transactivation, which is in accordance with the findings of Durand et al. (15). Interestingly, this receptor-AF2/{tau}c fusion (RAR-TR-AF2/{tau}c) exhibited silencing function in the absence of ligand in CV1 cells. This may be due to a stronger interaction of corepressors with this receptor fusion. Our electrophoretic mobility shift assay (EMSA) experiments indeed show an enhanced interaction of SMRT with the RAR-TR-AF2/{tau}c fusion. Similarily, mutations of AF2/{tau}c of RXR increased SMRT binding to the RAR/RXR heterodimer in EMSA experiments (30). Also, naturally occurring mutations of TRß, derived from patients with resistance to thyroid hormone with mutations outside of the characterized SMRT interaction region, also exhibit an unusually strong interaction with the corepressor SMRT (38).

Our data indicate that specifically the RAR-AF2/{tau}c sequence prevents silencing in the context of its receptor. This may indicate that despite sequence conservation, TR-derived AF2/{tau}c and RAR-derived AF2/{tau}c differ in their functionality in that AF2/{tau}c can have an influence on corepressor binding to the silencing domain in the absence of hormone.

Thus, amino acids of the conserved C-terminal activation domain AF2/{tau}c are required not only for corepressor release in the presence of hormone, but also have an influence on corepressor binding in the absence of hormone and consequently can influence silencing function of nuclear receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Expression plasmids coding for activation domain-Gal-fusions pABgal94 (9), pCMVgal-Oct (26), pCMVgal-p65 (26), pCMX-SMRT (6), and pCMX-N-CoR (7), and for Gal-hRAR{alpha} fusions pABgal147-RAR135–462 (5) and -RAR{Delta}C60 (5), have been described previously. pABgalRAR-AF2 was generated by an in-frame fusion of a synthetic oligonucleotide coding for RAR aa 403–419 into pABgal94. Gal-RAR403-TR445–461 (RAR-TR-AF2/{tau}c), Gal-RAR{Delta}C43, and pCMV-gal94RAR403-Oct and -RAR-p65 were cloned by introducing the coding sequences of RAR (aa 135–403) into the corresponding sites of pCMVGalOct and Galp65 (26) between the coding sequences of the Gal4 DNA-binding domain (DBD) and the activator sequence. The reporter plasmid p17mer tkCAT has been described previously (5).

Cell Culture
CV1 and L cells were grown in DMEM with 10% FCS at 37° C/5% CO2. Cotransfections were carried out using the CaPO4-method (27, 28) for CV1 cells and diethylaminoethyl-Dextran method (5) for L cells. Expression vector (0.5 pmol) was cotransfected with the 1.5 pmol of indicated reporter plasmid. Coexpression of SMRT was performed by addition of 1.75 pmol pCMX-SMRT (6) into the DNA transfer mix. For hormone treatment, cells were seeded out in charcoal-treated 10% FCS.

EMSA and Protein Source
Extracts from transfected COS cells (29, 41) were modified as follows. Transfected COS1 cells were harvested in PBS, pelleted, and resuspended in 5x packed cell volume binding buffer containing 20 mM HEPES, pH 7.8, 400 mM NaCl, 20% glycerol, and 2 mM dithiothreitol. Cells were lysed by freezing (-80 C) and thawing on ice. Cell debris were removed by centrifugation. EMSA was performed by preincubation of the protein extract with 1 µg deoxyinosinic-deoxycytidylic acid, 0.25 µg denatured calf thymus DNA in 0.5times] binding buffer. Bacterially expressed GST-SMRT (30) was purified and used (25–500 ng) according to Chen and Evans (6). After 10 min incubation on ice the 17 mer probe (5) was added and incubated for an additional 15 min on ice before loading a polyacrylamide gel, which was run with Tris/Glycine (25 mM/192 mM).

Western Analysis
Equal numbers of L or CV1 cells were lysed directly in SDS loading buffer and sonified for Western analysis. Goat anti-SMRT antibody directed against a peptide corresponding to aa 1477–1495 of SMRT C terminus (Santa Cruz Biotechnology) and anti-goat horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) was used for detection with the enhanced chemiluminescence analysis system (Amersham) according to the manufacturer’s protocol.


    ACKNOWLEDGMENTS
 
We thank Drs. A. Hörlein and M. G. Rosenfeld for pCMX-N-CoR and R. Evans for pCMX-SMRT. We are greatful to Kristine Krüger for excellent technical help. We thank Stephan Tenbaum and Dr. Claudia Baniahmad for critically reading the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Aria Baniahmad, Generisches Institut, Heinrich-Buff-Ring 58, Giessen, Germany D-35392.

This work was supported by grants from the Sonderforschungsbereich SFB 249 of the Deutsche Forschungsgemeinschaft and from the Fonds der Chemischen Industrie.

This work contains parts of the Ph.D. thesis of U. Dressel.

Received for publication October 20, 1997. Revision received January 8, 1998. Accepted for publication January 12, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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