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Endocrinology Vol. 147, No. 7 3408-3418
Copyright © 2006 by The Endocrine Society

Avian MyoD and c-Jun Coordinately Induce Transcriptional Activity of the 3,5,3'-Triiodothyronine Nuclear Receptor c-ErbA{alpha}1 in Proliferating Myoblasts

Muriel Busson1, Laetitia Daury1, Pascal Seyer, Stéphanie Grandemange, Laurence Pessemesse, François Casas, Chantal Wrutniak-Cabello and Gérard Cabello

Unité d’Endocrinologie Cellulaire, Unité Mixte de Recherche Différenciation Cellulaire et Croissance (Institut National de la Recherche Agonomique, Université Montpellier II, Ecole Nationale Supèrieure d’Agronomie de Montpellier), Institut National de la Recherche Agronomique, 34060 Montpellier, France

Address all correspondence and requests for reprints to: G. Cabello, Unité d’Endocrinologie Cellulaire, Unité Mixte de Recherche Différenciation Cellulaire et Croissance, Institut National de la Recherche Agronomique (INRA), 2 place Viala, 34060 Montpellier Cedex 1, France. E-mail: cabello{at}ensam.inra.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although physical interactions with other receptors have been reported, heterodimeric complexes of T3 nuclear receptors (TR) with retinoid X receptors (RXRs) are considered as major regulators of T3 target gene expression. However, despite the potent T3 influence in proliferating myoblasts, RXR isoforms are not expressed during proliferation, raising the question of the nature of the complex involved in TR{alpha} transcriptional activity. We have previously established that c-Jun induces TR{alpha}1 transcriptional activity in proliferating myoblasts not expressing RXR. This regulation is specific to the muscle lineage, suggesting the involvement of a muscle-specific factor. In this study, we found that MyoD expression in HeLa cells stimulates TR{alpha}1 activity, an influence potentiated by c-Jun coexpression. Similarly, in the absence of RXR, MyoD or c-Jun overexpression in myoblasts induces TR{alpha}1 transcriptional activity through a direct repeat 4 or an inverted palindrome 6 thyroid hormone response element. The highest rate of activity was recorded when c-Jun and MyoD were coexpressed. Using c-Jun-negative dominants, we established that MyoD influence on TR{alpha}1 activity needs c-Jun functionality. Furthermore, we demonstrated that TR{alpha}1 and MyoD physically interact in the hinge region of the receptor and the transactivation and basic helix loop helix domains of MyoD. RXR expression (spontaneously occurring at the onset of myoblast differentiation) in proliferating myoblasts abrogates these interactions. These data suggest that in the absence of RXR, TR{alpha}1 transcriptional activity in myoblasts is mediated through a complex including MyoD and c-Jun.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE IS considered as a major regulator of in vivo muscle development. It not only stimulates muscle growth by increasing the number and diameter of muscle fibers (1, 2), but it also regulates the transition between neonatal and adult myosin isoforms (3, 4) and the contractile features of adult muscle fibers (5). Despite this important influence, disruption of {alpha}- or ß-genes encoding thyroid hormone receptors detected in this tissue (6) only slightly affects muscle development in mice (7). This surprising observation could, in particular, be explained by the fact that thyroid hormone receptors repress T3 target gene expression when the hormone is absent; consequently, depletion of receptors may induce a less drastic phenotype (no stimulation of T3 target genes) than absence of the hormone does (repression of T3 target genes). However, given the lack of earlier in vitro studies, the mechanisms involved in the myogenic influence of T3 have only been gradually elucidated over the last decade by studies using secondary cultures of embryonic quail myoblasts or the avian myoblast line QM7. We first established that T3 accelerates irreversible myoblast withdrawal from the cell cycle and consequently potentiates myoblast differentiation (8). These data clearly suggest that the major myogenic influence of this hormone targets proliferating myoblasts. Study of the molecular events involved in this regulation indicates that, whereas T3 stimulates MyoD and Myogenin expression in murine myoblasts (9), a major and more general mechanism occurring in several species is probably the repression of transcriptional activity in the c-Jun/c-Fos activator protein 1 (AP-1) complex, which is considered as a strong repressor of myoblast differentiation (10).

As our study of molecular mechanisms underlying the myogenic influence of T3 in avian myoblasts progressed, we observed that, owing to the lack of retinoid X receptor (RXR) expression (11), T3 nuclear receptor {alpha}1 (TR{alpha}1)/RXR complexes do not occur in proliferating myoblasts. Downes et al. (12) have reported similar data in murine C2C12 myoblasts and provided evidence that RXR isoform expression is only induced at the onset of terminal myoblast differentiation. In agreement with these results, TR{alpha}1 did not display any significant transcriptional activity in these cells through a direct repeat 4 thyroid response element (DR4-TRE) in transient transfection experiments using reporter genes, although RXR expression by transient or stable transfection restored its transcriptional activity in the presence of T3. Because myogenic T3 influence is induced during the myoblasts’ proliferation period, these results raised the question of TR{alpha} transcriptional activity in proliferating cells. Interestingly, we found that c-Jun expression induced TR{alpha}1 activity through a DR4-TRE. But this surprising c-Jun influence did not occur in HeLa cells, thus suggesting that the functional interaction between TR{alpha}1 and the cellular oncogene requires the occurrence of a muscle-specific protein (muscle-specific factor, MSF) (11).

More recently, we found that TR{alpha}1 overexpression fully abrogates avian MyoD transcriptional activity, thus highlighting the occurrence of a functional interaction between TR{alpha}1 and the muscle-specific factor MyoD (13). Therefore, the purpose of this study was to test the possibility that, in proliferating myoblasts not expressing RXR, TR{alpha} transcriptional activity is at least partly induced through muscle-specific interactions with c-Jun and MyoD. In this study, we found that in myoblasts, MyoD induces TR{alpha}1 transcriptional activity through DR4 and inverted palindrome (Ipal)-TREs in a c-Jun-dependent manner. This functional interaction involves physical interactions and is abrogated by RXR expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Quail myoblasts of the QM7 cell line (14) were seeded at a plating density of 7000 cells/cm2. They were grown in Earle’s 199 medium supplemented with tryptose phosphate broth (0.2%), L-glutamine (2 mM), gentamycin (50 µg/ml), and fetal calf serum (10% vol/vol). Serum was T3 depleted according to Samuels et al. (15). After hormonal depletion, serum T4 and T3 concentrations measured by RIA were below the limit of detection in the assay.

Plasmid and reporter genes
The DR4-tk-CAT plasmid upstream of the thymidine kinase promoter linked to the CAT gene was kindly provided by P. Chambon (LGME U184, Strasbourg, France). The gal4-tk-CAT reporter plasmid contains four response elements of the yeast transcription factor GAL4 upstream of the chloramphenicol acetyltransferase (CAT) coding sequence. The myogenin-CAT reporter plasmid contains the –131/+40 fragment of the chicken myogenin promoter upstream of the CAT coding sequence (16). The –73col CAT collagenase promoter has been described elsewhere (17). The expression vectors for chicken TR{alpha}1 and MyoD (pRSV TR{alpha}1 and pRSV CMD1) have already been described (18, 19). The RSV (Rous sarcoma virus)-ß-galactosidase expression vector has been described by Cassar-Malek et al. (11). The expression vector for rat Gal4/TR{alpha}1 (pSVGal4{alpha}1) was provided by Dr. F. Flamant (ENS, Lyon, France).

The expression vector for quail c-Jun (pDP18 c-Jun) and chicken RXR{gamma} (pRSV RXR{gamma}) have already been described (20, 21). The pDP18 c-Jun D176 expression vector (22) led to the expression of a quail c-Jun mutant deleted from almost all the transactivating domain. The pDP18 c-Jun eb1 expression vector (22) led to the expression of a quail c-Jun mutant in which the natural dimerization domain was replaced by the dimerization domain of the viral EB1 protein (c-Jun eb1 forms only homodimers).

pGEM-T TR{alpha}1, encoding the full-length thyroid hormone receptor TR{alpha}1, pGEM-T TR{alpha}1 (36–410), and pGEM-T TR{alpha}1 (152–410) have been described by Casas et al. (23). pGEM-T MyoD, pGEM-T JUN, and the mutants of TR{alpha}1 were generated by PCR from the full-length corresponding mRNA, inserted into the EcoRV site of pGEM-T plasmid (Promega, Charbonnieres, France).

Mutants of avian MyoD were generated with specific primers from the plasmid pRSV CMD1 and inserted in the SalI/NotI sites of the pGEX4T2 (Pharmacia, Uppsala, Sweden) vector, in frame with the glutathione-S-transferase (GST) coding sequence.

Transient transfections and CAT assays
Transient transfections were performed using the calcium phosphate coprecipitation procedure as described by Cassar-Malek et al. (11). One microgram of pCMV ß-galactosidase expression vector was cotransfected to provide an internal control of transfection efficiency. After cell exposure to precipitates for 24 h, the DNA-containing medium was replaced with fresh medium containing T3 (10–8 M) when indicated, and the cells were grown for another 24 h. ß-Galactosidase activity was measured as previously described (24). CAT enzymatic activity was measured by following the kinetics of chloramphenicol acetylation (11). For each assay, the initial rate of the enzymatic reaction was determined, and results were expressed as a percentage of control values after ß-galactosidase normalization.

In vitro transcription and translation
In vitro transcription and translation were carried out with TNT-coupled reticulocyte lysate system (Promega) and trans-[35S]methionine (ICN, Costa Mesa, CA), according to the manufacturer’s protocol. The proteins were processed by SDS-PAGE, and quantification of the relative intensities was carried out with a phosphoimager (Molecular Dynamics, Sunnyvale, CA).

Western blots
Fifty micrograms of proteins of cell lysates were resolved by 10% SDS-PAGE minigels and blotted onto polyvinylidene difluoride membranes that were further incubated with anti-c-Jun (kindly provided by Dr. M. Castellazzi, Lyon, France), anti-MyoD (kindly provided by Dr. B. M. Paterson, Seattle, WA), anti-TR{alpha}1 RHTII (25), or anti-RXR (kindly provided by Prof. P. Chambon, Strasbourg, France). Signals were further detected by enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK) using a second horseradish peroxidase-conjugated antibody provided by the manufacturer.

Coimmunoprecipitation
Immunoprecipitation of proteins was performed as described by Casas et al. (26), with a few modifications, with in vitro-transcripted and -translated 35S-labeled avian MyoD, RXR, c-Jun, TR{alpha}1, and mutants of avian MyoD and TR{alpha}1. Ten microliters of each protein were mixed with 1 µl of antibody raised against TR{alpha}1 or MyoD and 300 µl of binding buffer [50 mM HEPES (pH 7), 400 mM NaCl, 1% Nonidet P-40, and 1 µg/ml aprotinin] overnight at 4 C. Samples were incubated with 30 µl of protein G-Sepharose for 3 h at 4 C and washed three times with binding buffer and three times with TNE buffer [10 mM Tris (pH 7.5), 150 mM NaCl, and 1.2 mM EDTA]. Bound proteins were separated by SDS-PAGE, and the dried gel was exposed for autoradiography and analyzed with a phosphoimager (Molecular Dynamics).

A similar procedure was used for immunoprecipitation of cell extracts, using 400 µg cell lysate proteins and 2 µl of antibody raised against c-Jun. Revelation was performed as indicated for Western blotting with an antibody raised against TR{alpha}1. However, in HeLa cells, the second antibody used for enhanced chemiluminescence was rabbit TrueBlot (Cliniscience Laboratories, Montrouge, France), not recognizing Igs.

GST pull-down experiments (27)
GST and GST fusion proteins were expressed in Escherichia coli DH5{alpha}, purified on glutathione-Sepharose beads (Amersham) and quantified by the Bradford method (Bio-Rad protein assay). Twenty micrograms of the GST fusion proteins were processed for SDS-PAGE and Coomassie stained to confirm the integrity of the full-length fusion proteins and to determine the amounts to use for the pull-down experiment.

For in vitro protein-protein interaction assays, comparable amounts of GST fusion proteins beads were incubated with 5 µl of in vitro-translated proteins in binding buffer [20 mM HEPES (pH 7.9), 50 mM KCl, 2.5 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin] overnight at 4 C. After five washes in RIPA buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin), the bound proteins were separated by SDS-PAGE; the gel was then dried for 1 h at 70 C, exposed for autoradiography, and analyzed with a phosphoimager (Molecular Dynamics).

Statistical analysis
Statistical analyses were performed using the paired t test (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
c-Jun expression restores TR{alpha}1 transcriptional activity in proliferating myoblasts
We have previously shown that, in the absence of RXR, AP-1 activity does not inhibit TR{alpha}1 transcriptional activity. Furthermore, we reported that, in these circumstances, c-Jun expression is able to induce T3-dependent transcriptional activity in the TR{alpha}1 through a DR4-TRE (11). To confirm and extend these data to other TREs, we performed transient transfection experiments using CAT reporter genes under the control of DR4-, palindromic (pal)-, or Ipal-TREs. Protein levels obtained after overexpression are shown in Fig. 1AGo. Whereas TR{alpha} by itself displayed a T3-dependent transcriptional activity only through a pal-TRE (Fig. 1Go, C–E), coexpression of c-Jun restored positive TR{alpha} activity when reporter genes under the control of a DR4- or an Ipal6-TRE were used (respectively, 5- and 4-fold stimulation of CAT activity; P < 0.001 and P < 0.005; Fig. 1Go, D and E). However, it did not affect specific TR{alpha} activity through a pal-TRE (Fig. 1CGo). In parallel experiments, we found in myoblasts that RXR{gamma} coexpression induced a positive TR{alpha} transcriptional activity only through a DR4-TRE (data not shown).


Figure 1
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  		FIG. 1. Influence of c-Jun on TR{alpha}1 transcriptional activity in myoblasts. A, Sequences of the three TREs used in all experiments; B–E, QM7 myoblasts were transfected with 1 µg/dish of the reporter TRE pal-Glo-CAT (B), DR4-tk-CAT (C), or Ipal6-tk-CAT (D) and 2 µg of TR{alpha}1 and/or c-Jun expression vectors in the absence or presence of T3 (10 nM). In all cases, the amount of transfected DNA was equalized by addition of the same empty vectors that were used for the cotransfected factors. All results are expressed as a percentage of CAT activity measured in basal conditions after ß-galactosidase normalization. Data are presented as the mean ± SEM of five separate experiments. **, P < 0.01; ***, P < 0.005 when compared with control cells without T3 stimulation. E, Western blot experiments indicating the levels of TR{alpha}1 and c-Jun reached after transient overexpression of each factor: 1, empty vectors; 2, TR{alpha}1; 3, c-Jun; and 4, TR{alpha}1 plus c-Jun. These transfections do not induce RXR expression.

 
Influence of c-Jun and MyoD on TR{alpha}1 transcriptional activity
Taking into account the induction of TR{alpha}1 transcriptional activity through a DR4- or Ipal-TRE observed in the previous experiment, we tested the cell specificity of this functional interaction. To this end, transient transfection experiments were performed in HeLa cells and in QM7 myoblasts. Protein levels obtained after overexpression are shown in Fig. 2AGo.


Figure 2
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  		FIG. 2. c-Jun and MyoD potentiate TR{alpha}1 transcriptional activity in HeLa cells and in QM7 myoblasts but does not induce RXR expression. HeLa cells (A) or QM7 myoblasts (B) were transfected with 1 µg/dish of Ipal6-tk-CAT reporter gene and 2 µg TR{alpha}1, c-Jun, and/or MyoD expression vectors in the absence or presence of T3 (10 nM). In all cases, the amount of transfected DNA was equalized by addition of the same empty vectors that were used for the cotransfected factors. All results are expressed as a percentage of CAT activity measured in basal conditions after ß-galactosidase normalization. Data are presented as the mean ± SEM of four separate experiments. **, P < 0.01; ***, P < 0.005 when compared with control cells without T3 stimulation. °, P < 0.05; °°, P < 0.01; °°°, P < 0.005 when compared with TR{alpha}1 overexpressing cells in the presence of T3. {ddagger}, P < 0.05 when compared with TR{alpha}1- plus c-Jun-overexpressing cells in the presence of T3. Data obtained in TR{alpha}1- plus MyoD-overexpressing cells were not significantly different from that obtained in TR{alpha}1- plus c-Jun- or TR{alpha}1- plus c-Jun- plus MyoD-overexpressing cells (P > 0.05). C, Western blot experiments indicating the levels of TR{alpha}1, MyoD, and c-Jun reached after transient overexpression of each factor: 1, empty vectors; 2, TR{alpha}1; 3, c-Jun; 4, MyoD; 5, TR{alpha}1 plus c-Jun; 6, TR{alpha}1 plus MyoD; and 7, TR{alpha}1 plus c-Jun plus MyoD. D, Westerns blot experiments indicating that c-Jun and/or MyoD overexpression does not induce RXR expression: 1, empty vectors; 2, c-Jun; 3, MyoD; 4, c-Jun plus MyoD; 5, empty lane; and 6, in vitro synthesized RXR{alpha} and RXR{gamma} proteins (reticulocyte lysate, vol/vol).

 
As expected, TR{alpha}1 displayed significant transcriptional activity through an Ipal-TRE in HeLa cells, which naturally express RXR isoforms (3-fold increase; P < 0.01; Fig. 2BGo). However, c-Jun expression did not significantly change the extent of this transcriptional stimulation (3.1-fold increase compared with the control level). We tested the possibility that this c-Jun inefficiency resulted from the absence of a muscle-specific factor. Interestingly, we observed that avian MyoD expression on its own stimulated ligand-dependent transcriptional activity of TR{alpha}1 (2-fold increase; P < 0.01; Fig. 2BGo). In addition, the highest rate of activity was recorded when c-Jun and MyoD were simultaneously coexpressed with TR{alpha} (Fig. 2BGo; 2.5-fold increase compared with TR{alpha} alone; P < 0.005).

Similar experiments were performed on QM7 myoblasts using an Ipal6-TRE. c-Jun or MyoD coexpression induced significant ligand-dependent TR{alpha} activity through this TRE, but again the highest activity was recorded in the presence of the three proteins (Fig. 2CGo). Moreover, this TR{alpha}1 transcriptional activity did not result from the induction of RXR{alpha} or -{gamma} expression by c-Jun and/or MyoD expression (Fig. 2DGo).

To study these functional interactions further without any interference by endogenous nuclear receptors able to heterodimerize with TR{alpha}, we performed similar experiments using the Gal4 system (gal4-tk-CAT reporter and a chimeric T3 receptor including the DNA-binding domain of Gal4 fused to the hinge region and the ligand-binding domain of TR{alpha}1). In these conditions, homodimers of the chimeric T3 receptor stimulate transcription of the reporter gene, as shown in Fig. 3Go. Even using this experimental model, MyoD and c-Jun moderately but significantly stimulated Gal4-TR{alpha}1 ligand-dependent activity (P < 0.05 and P < 0.01, respectively). In addition, coexpression of Gal4-TR{alpha}1, c-Jun, and MyoD induced a sharp rise in the transcriptional activity of the chimeric receptor (2.5-fold increase; P < 0.001; Fig. 3Go).


Figure 3
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  		FIG. 3. c-Jun and MyoD potentiation of Gal4/TR{alpha}1 chimera receptor transcriptional activity in myoblasts needs the transactivation and dimerization domains of c-Jun. QM7 myoblasts were transfected with 1 µg/dish of Gal4-tk-CAT reporter gene and 2 µg Gal4/TR{alpha}1, MyoD, and/or c-Jun, c-Jun {Delta}176, c-Jun eb1 expression vectors in the absence or presence of T3 (10 nM). In all cases, the amount of transfected DNA was equalized by addition of the same empty vectors that were used for the cotransfected factors. Results are expressed as percentage of CAT activity in basal conditions, after ß-galactosidase normalization. Data are presented as the mean ± SEM of three separate experiments. ***, P < 0.005 compared with control cells without T3 stimulation; °, P < 0.05; °°°, P < 0.005 compared with TR{alpha}1-overexpressing cells in the presence of T3; {ddagger}, P > 0.01 compared with TR{alpha}1- plus c-Jun-overexpressing cells in the presence of T3; #, P < 0.01 compared with TR{alpha}1- plus MyoD-overexpressing cells in the presence of T3.

 
Furthermore, we have overexpressed two c-Jun mutants unable to functionally interact with MyoD instead of c-Jun. In c-Jun eb1, the natural dimerization domain has been replaced by a sequence allowing only homodimerization of the mutant (22); c-Jun{Delta}176 is deleted from the transactivation domain of the protein. These two domains have been shown to be of crucial importance for interaction with MyoD (29). In addition, c-Jun{Delta}176 could also act as a dominant negative factor relatively to endogenous c-Jun. Interestingly, c-Jun mutants that can form only homodimers (c-Jun eb1), or which are deleted from the transactivation domain (c-Jun{Delta}176), did not affect Gal4-TR{alpha}1 activity, unlike WT-c-Jun (Fig. 3Go). Furthermore, coexpression of these mutants fully abrogated the ability of MyoD to stimulate the transcriptional activity of the chimeric receptor (Fig. 3Go), thus suggesting that restoration (TR{alpha}) or stimulation (Gal4-TR{alpha}1) of receptor functionality results from interactions between the three partners. In addition, these data led us to conclude that the natural NH2 terminus and DNA-binding domains of TR{alpha}1 are not involved in these functional interactions.

Physical interaction between MyoD and TR{alpha}1
Physical interactions between MyoD and c-Jun have already been identified and involve the same domains of the cellular oncoprotein as those shown to be crucial for the restoration of TR{alpha} transcriptional activity (transactivation and dimerization domains of the cellular oncogene) (29). We therefore tested the possibility of the occurrence of an additional physical interaction between MyoD and TR{alpha} leading to indirect recruitment of c-Jun by TR{alpha}.

In our first approach, we used coimmunoprecipitation experiments with in vitro-synthesized proteins. In these assays, MyoD and GFP were 35S radiolabeled. Although devoid of any interaction with MyoD alone (Fig. 4AGo, lane 2), the antibody raised against the TR{alpha}-NH2 terminus immunoprecipitated the myogenic factor only when TR{alpha}1 was present in the reaction mix (Fig. 4AGo, lane 3). Failure to immunoprecipitate GFP underlined the specificity of the interaction (Fig. 4Go, lane 1). This result was reassessed in GST pull-down experiments (Fig. 4BGo). As expected, the 35S-radiolabeled T3 receptor did not interact with GST alone (Fig. 4BGo, lane 2) but was retained by GST-MyoD beads (Fig. 4BGo, lane 4). The 35S-radiolabeled GFP protein, used as a negative control, did not interact with GST or GST-MyoD (Fig. 4BGo, lanes 1 and 3). Similar experiments performed in the presence of increasing amounts of T3 demonstrated that the interaction was independent of the ligand presence: T3 addition (10–8 and 10–7 M) did not influence MyoD/TR interaction (Fig. 4CGo). Lastly, to test the occurrence of this interaction in living cells, we performed coimmunoprecipitation experiments in QM7 myoblasts. Because endogenous TR{alpha}1 is frequently at the brink of detection in these cells (Fig. 1EGo), we overexpressed this receptor by transient transfection, and we used antibodies raised against c-Jun to immunoprecipitate this protein. In agreement with our previous data, these experiments demonstrated that TR{alpha}1 interacts with c-Jun in MyoD-expressing cells (Fig. 4DGo). Moreover, using the same approach in HeLa cells, which do not express MyoD, we found that an antibody raised against c-Jun immunoprecipitates TR{alpha}1 only after MyoD expression (Fig. 4EGo). This last result confirms that the TR{alpha}1/c-Jun physical interaction needs MyoD recruitment by the T3 receptor.


Figure 4
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  		FIG. 4. TR and MyoD proteins physically interact directly regardless of the presence of T3. A, Coimmunoprecipitation experiment using an antibody raised against TR and radiolabeled in vitro-synthesized [35S]GFP and [35S]MyoD proteins and in vitro-synthesized cold TR. B, GST pull-down assay using in vitro-synthesized [35S]TR{alpha}1 or [35S]GFP (negative control) proteins and GST-MyoD or GST beads. The input corresponds to one third of the amount of in vitro-translated products loaded in each lane. C, GST pull-down assay using in vitro-synthesized [35S]TR{alpha}1 proteins and GST-MyoD or GST beads in the presence of different amounts of T3. The input corresponds to one third of the amount of in vitro-translated TR loaded in each lane. All these results are representative of at least three separate experiments for each interaction. D, In QM7 myoblasts, overexpressed TR{alpha}1 coimmunoprecipitates with endogenous c-Jun in QM7 myoblasts. Immunoprecipitation was performed with a c-Jun antibody. Western blot was performed with a TR{alpha} antibody. 1, Control cells; 2 and 3, overexpression of TR{alpha}1. E, In HeLa cells, overexpressed TR{alpha}1 coimmunoprecipitates with endogenous c-Jun only in the presence of MyoD. Immunoprecipitation was performed with a c-Jun antibody. Western blot was performed with a TR{alpha} antibody. 1, TR{alpha}1 overexpression (5 µg expression vector); 2, TR{alpha}1 (5 µg) and MyoD overexpression (1 µg expression vector); 3, TR{alpha}1 (5 µg) and MyoD overexpression (2 µg expression vector); 4, TR{alpha}1 (5 µg) and MyoD overexpression (5 µg expression vector). In contrast to D, the second antibody (coupled to peroxidase) does not recognize Ig (see Materials and Methods). Two exposure times of the same blot are shown.

 
Characterization of TR{alpha} domains involved in this interaction
With the aim of characterizing the TR{alpha} domains involved in the interaction with MyoD, we used several deletion mutants of the receptor (Fig. 5AGo) in GST pull-down experiments. All TR{alpha}1 mutants sharing the DNA-binding domain and the hinge region strongly interacted with GST-MyoD (Fig. 5CGo). Results obtained with the 1–408, 36–408, 36–194, and 152–408 mutants demonstrated that the 36–152 amino acid sequence plays an important role in TR{alpha}1 interaction with the myogenic factor; this is supported by the use of the 1–194 mutant (Fig. 5CGo). In addition, the carboxyl-terminal part of the receptor did not significantly interact with MyoD (152–408 and 194–408 mutants, Fig. 5CGo).


Figure 5
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  		FIG. 5. The 36–152 amino acid sequence of TR induces interaction with MyoD. A, Schematic diagram of TR{alpha}1 mutants used in this experiment. Interaction efficiency of TR{alpha}1 wild-type or mutants with MyoD is shown on the right of the diagram. B, SDS-PAGE showing the synthesis efficiency of each 35S-labeled TR{alpha}1 mutant. Note that translation efficiency was very poor for the 36–194 mutant. C, GST pull-down assay using in vitro-synthesized 35S-labeled TR{alpha}1 mutants with GST-MyoD or GST beads. Because of the lower amounts of the 36–194 mutant, the time of exposure was longer than for other mutants. These results are representative of three separate experiments.

 
When compared with the functional study using the chimeric Gal4-TR, demonstrating that the natural DNA-binding domain of TR{alpha}1 is not needed for the interaction with MyoD (Fig. 3Go), these data indicate that the 121–152 hinge region of the receptor is of crucial importance for binding to the myogenic factor.

Characterization of MyoD domains involved in this interaction
Using a similar experimental approach, we attempted to characterize the MyoD domains involved in the interaction with TR{alpha}1 in GST pull-down experiments. To this end, we used several mutants of the myogenic factor fused to GST (Fig. 6AGo). We observed first that only one mutant did not interact with TR{alpha}1 (Fig. 6BGo, 162–318 mutant), thus ruling out involvement by the carboxyl-terminal part of MyoD. Assessment of the results obtained with 1–162 and 52–318 mutants led us to conclude that the 52–162 domain interacts with TR{alpha}1 (Fig. 6BGo). More specifically, using the 102–162 deletion mutant, we found that the basic helix loop helix (bHLH) domain of MyoD is involved in the interaction with TR{alpha}1 (Fig. 6BGo). However, a second interaction sequence was also observed in the transactivation domain (1–52 sequence, Fig. 6BGo) of the myogenic factor.


Figure 6
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  		FIG. 6. The transactivation and bHLH domains of MyoD induce interaction with TR. A, Left, schematic diagram of all MyoD mutants with a summary of the interaction efficiency between TR and MyoD wild-type or mutants obtained in B (TAD, transactivation domain; BHLH, bHLH domain); right, synthesis efficiency of each GST-MyoD mutant was assessed by Coomassie-stained SDS-PAGE. *, GST-MyoD signal. Signal intensity was used to load a similar amount of each mutant in GST pull-down experiments. B, GST pull-down assay using in vitro-synthesized 35S-labeled TR{alpha}1 with GST-MyoD mutants or GST beads. These results are representative of four separate experiments.

 
RXR{gamma} abrogates TR{alpha}1/c-Jun/MyoD functional interactions
To study the influence of the induction of RXR expression occurring at the onset of myoblast differentiation, we tested its influence on the ability of TR{alpha}1 to interact functionally with MyoD and c-Jun.

This study was performed in transient transfection experiments with the Gal4 system previously used to avoid interferences by endogenous nuclear receptors able to heterodimerize with TR{alpha}. As expected, RXR{gamma} expression did not influence basal Gal4-tk-CAT reporter expression (Fig. 7AGo) and did not affect Gal4-TR{alpha}1 transcriptional activity independently of the presence of T3 (Fig. 7BGo). More interesting is the observation that in the presence of RXR{gamma}, c-Jun (58-fold stimulation of reporter activity), MyoD (54-fold stimulation), and c-Jun plus MyoD (62-fold stimulation) did not stimulate Gal4-TR{alpha}1 transcriptional activity (60-fold stimulation; not significant; Fig. 7BGo).


Figure 7
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  		FIG. 7. RXR{gamma} expression abrogates the functionality of Gal4/TR{alpha}1/c-Jun/MyoD interaction in myoblasts. A, QM7 myoblasts were transfected with 1 µg/dish of Gal4-tk-CAT reporter gene and 2 µg RXR{gamma} and/or MyoD or c-Jun expression vectors. In all cases, the amount of transfected DNA was equalized by addition of the same empty vectors that were used for the cotransfected factors. Results are expressed as a percentage of CAT activity measured in basal conditions after ß-galactosidase normalization. Data are presented as the mean ± SEM of three separate experiments. ***, P < 0.005 when compared with control cells without T3 stimulation. B, QM7 myoblasts were transfected with 1 µg/dish of Gal4-tk-CAT reporter gene and 2 µg of Gal4/TR{alpha}1, MyoD, c-Jun, and/or RXR{gamma} expression vectors. Results are expressed as a percentage of CAT activity measured in basal conditions after ß-galactosidase normalization. Data are presented as the mean ± SEM of three separate experiments. ***, P < 0.005 when compared with control cells without T3 stimulation; °, P < 0.05; °°°, P < 0.005 when compared with TR{alpha}1-overexpressing cells in the presence of T3. C, Western blot experiments indicating the levels of RXR{gamma}, Gal4-TR{alpha}1, MyoD, and c-Jun reached after transient overexpression of each factor: 1, Gal4-TR{alpha} plus c-Jun; 2, Gal4-TR{alpha} plus MyoD; 3, Gal4-TR{alpha} plus c-Jun plus MyoD; 4, Gal4-TR{alpha} plus RXR{gamma}; 5, Gal4-TR{alpha} plus c-Jun plus RXR{gamma}; 6, Gal4-TR{alpha} plus MyoD plus RXR{gamma}; and 7, Gal4-TR{alpha} plus c-Jun plus MyoD plus RXR{gamma}.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies have been performed to define the importance of RXR for TR{alpha} transcriptional activity. Sugawara et al. (30) reported that TR{alpha}/RXR are quantitatively predominant in all transcriptional complexes including TRs. In addition, several in vitro experiments suggest that T3 dissociates TR{alpha} homodimers from DR4-TREs but does not affect this binding to a pal-TRE or the binding of TR{alpha}/RXR complexes to DR4-TREs (31, 32, 33). These observations gave rise to the idea that TR{alpha}/RXR is an important T3-dependent transcription complex, at least through a DR4-TRE (34, 35). More generally, TR/RXR are considered as major complexes for the control of gene expression by the thyroid hormone. However, RXR expression is obviously different from one cell type to another; for instance, TR/RXR complexes are not detectable in ES cells because of the very low expression levels of RXR (36). Moreover, two separate studies have reported that RXR isoforms are not expressed in mice or avian proliferating myoblasts (11, 12). In the light of the observation that T3 stimulates myogenic differentiation by acting on proliferating myoblasts and inducing their withdrawal from the cell cycle (8, 37), these data clearly suggested that T3 transcriptional activity in myoblasts is mediated through TR complexes that do not include RXR.

TR/c-Jun/MyoD functional interactions in myoblasts
c-Jun is generally considered as a potent repressor of TR transcriptional activity through its involvement in the c-Jun/c-Fos AP-1 complex (38, 39). However, we have previously reported that, in the absence of RXR, AP-1 activity (c-Jun/c-Fos) does not repress TR{alpha}1 transcriptional activity in myoblasts. Instead of this, we found that, in these circumstances, c-Jun expression induces positive TR{alpha}1 activity through a DR4-TRE (11), a result extended in the present study to the Ipal-TRE. In addition, as observed for RXR (data not shown), this influence is not detected using a reporter gene controlled by a pal-TRE. Overall, this set of data suggests that, in proliferating myoblasts, transcriptional activity of TR{alpha}1 is mediated by homodimers or TR{alpha}/retinoic acid receptor complexes (pal-TRE) or by functional interactions with c-Jun (DR4- and Ipal-TREs).

As expected (38, 39), we found in our initial study that, in nonmyogenic cells expressing RXR (HeLa cells), c-Jun expression efficiently represses TR{alpha}1 transcriptional activity through pal- and DR4-TREs (11). In the present study, we report that this repression is not efficient through an Ipal-TRE, a regulatory sequence not previously used to study TR{alpha}/AP-1 antagonism. Interestingly, it appears that using this particular TRE, which is insensitive to the functional TR{alpha}/AP-1 antagonism, c-Jun does not stimulate TR{alpha}1 transcriptional activity in HeLa cells. This failure could result from the absence of a muscle-specific partner. This possibility is well supported by the observation that MyoD expression stimulates TR{alpha} transcriptional activity in these cells and that the highest rate of activity is recorded when TR{alpha}, c-Jun, and MyoD are expressed simultaneously. Furthermore, using the Gal4 system working only with Gal4-TR homodimers in myoblasts, we observed that c-Jun or MyoD stimulates the activity of the chimeric receptor and that the greatest stimulation occurred again in the presence of c-Jun and MyoD. However, we also observed that this influence of c-Jun and MyoD was not related to the induction of RXR expression. In addition, after having established that c-Jun mutants lacking the transactivation or the natural dimerization domains are unable to stimulate TR{alpha} activity, we found that they fully abrogate the influence of MyoD, thus suggesting that functionality of the interaction requires the presence of both c-Jun and MyoD.

TR/MyoD/c-Jun physical interactions
Several arguments stress the occurrence of a common physical interaction between TR{alpha}, MyoD, and c-Jun. The myogenic factor has already been shown to interact physically with RXR, a member of the nuclear receptor superfamily belonging to the same subclass as TR. In the present study, coimmunoprecipitation and GST pull-down experiments clearly support the existence of direct physical interactions between TR{alpha} and MyoD and have identified the interacting sequences of each partner. Despite the fact that, with one exception (40), all teams working on TR/AP-1 interactions have failed to detect physical interactions between TR and c-Jun, MyoD and c-Jun do physically interact (29). Furthermore, in coimmunoprecipitation experiments, we report the occurrence of a TR{alpha}/c-Jun physical interaction in myoblasts. However, in HeLa cells that do not express MyoD, this interaction does not occur but is induced by expression of different amounts of MyoD. This set of results led us to propose that interaction of TR{alpha} with MyoD enables the recruitment of c-Jun in a complex inducing TR{alpha} activity through DR4- and Ipal-TREs.

Interestingly, like TR{alpha}, c-Jun represses MyoD transcriptional activity (29). Furthermore, the transactivation and dimerization domains of c-Jun play a crucial role in MyoD/c-Jun interaction. In the present study, we have established that the same c-Jun domains are essential for the stimulation of TR{alpha} activity by the cellular oncoprotein and by MyoD. All these data could be summarized as follows: 1) TR{alpha} and c-Jun similarly repress MyoD activity; 2) c-Jun and MyoD similarly stimulate TR{alpha} activity; 3) c-Jun stimulates TR{alpha} activity only in the presence of MyoD, and MyoD stimulatory activity is abrogated by mutated or deleted forms of c-Jun unable to interact with the myogenic factor; 4) identical c-Jun domains are involved in the repression of MyoD transcriptional activity and in the stimulation of TR{alpha} activity; and 5) TR{alpha} physically interacts with MyoD and MyoD physically interacts with c-Jun. This set of results led us to propose that interaction of TR{alpha} with MyoD enables the recruitment of c-Jun in a complex inducing TR{alpha} activity through DR4- and Ipal-TREs and repressing MyoD activity through an E box (Fig. 8Go). This possibility is well supported by the observation that, in HeLa cells, TR{alpha} is immunoprecipitated by a c-Jun antibody only when MyoD is expressed.


Figure 8
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  		FIG. 8. Hypothesis concerning the influence of a TR/MyoD/c-Jun complex on TR and MyoD transcriptional activity. RXR isoforms are not expressed in proliferating myoblasts (12 ). During this period, TR physically interacts with MyoD, enabling the recruitment of c-Jun in a transcriptional complex active on TRE sequences (Ipal- or DR4-TREs) but inactive on E boxes (sequences recognized by the myogenic factor MyoD). This interaction induces TR transcriptional activity but represses the activity of MyoD and therefore could delay the onset of myoblast differentiation. During terminal differentiation, TR/RXR complexes replace the TR/MyoD/c-Jun complex with different consequences for the regulation of T3 target gene expression because of the nature of the TRE in the promoter of the gene. Disruption of the TR/MyoD/c-Jun complex allows MyoD to interact with its heterodimerization partner E12, to bind to its cognate response element (E box), and to stimulate the expression of genes involved in myogenic differentiation.

 
RXR expression abrogates the functionality of TR{alpha}/MyoD/c-Jun interaction
Because RXR expression occurs at the onset of myoblast differentiation (12), we studied the influence of RXR on the functionality of TR{alpha}/MyoD/c-Jun interaction. Using the Gal4 system in which transcription can be only stimulated by homodimeric binding of the chimeric Gal4-T3 receptor, we established that RXR abrogates the ability of c-Jun, MyoD, or c-Jun plus MyoD to enhance TR{alpha}1 transcriptional activity. In this model, RXR cannot bind to DNA in heterodimeric complexes with Gal4-TR, thus suggesting that this influence probably results from recruitment of MyoD by RXR in a previously identified RXR/MyoD complex (41). However, on natural TREs, TR/MyoD/c-Jun complexes could be replaced by the major TR/RXR complex, as suggested by restoration of this heterodimeric complex in electrophoretic mobility shift assay experiments after RXR overexpression in myoblasts (11). Therefore, this possibility raises the question of the control of T3 target genes driven by an Ipal-TRE, which is not sensitive to TR/RXR complexes (data not shown). Interestingly, if physiologically relevant, this event could lead to differential stimulation of gene expression by T3 in proliferating (functionality of all TREs) and differentiating (Ipal-TRE not activated) myoblasts, inducing a duality in the regulation of myoblast differentiation by TRs. Such duality is already apparent in the observation that TR{alpha}1 inhibits MyoD activity in proliferating myoblasts (13), thus repressing terminal differentiation, but represses AP-1 activity (a major inhibitor of myogenic differentiation) at the onset of terminal differentiation. Interestingly, we have reported a similar duality of c-Jun influence during myoblast proliferation (repression of myoblast withdrawal from the cell cycle) and differentiation (stimulation of myogenin expression and myoblast differentiation) (10). These data are in agreement with a recent work in progress of our team using microarray analysis, clearly suggesting that T3 target genes significantly differ in proliferating myoblasts when compared with differentiating cells (in particular, genes involved in withdrawal from the cell cycle vs. genes involved in differentiation).

In conclusion, although their exact physiological relevance remains to be more clearly established, the present data indicate that RXR is probably an important, but not essential, partner involved in the transcriptional activity of the T3 receptor. Instead of this, they highlight the possible existence of cell-specific complexes able to support TR transcriptional activity, thus introducing an additional level of regulation resulting in fine tuning of T3 influence during cell proliferation and differentiation, at least in the myogenic lineage (Fig. 8Go). In particular, we propose that in proliferating myoblasts, TR transcriptional activity is induced through a TR/MyoD/c-Jun complex, thus reducing the amounts of the myogenic factors able to bind to their specific response elements and delaying the onset of myoblast differentiation. Thereafter, RXR expression occurring at the induction of terminal differentiation could disrupt this complex, thus increasing the amounts of MyoD able to bind to its response element and to stimulate terminal differentiation. Such a mechanism could partly explain the prominent influence of T3 on muscle development by increasing the duration of the proliferative period of myoblasts and stimulating terminal differentiation.


   Acknowledgments
 
We are grateful to Dr. Samarut, Dr. Flamant, Dr. Rouault (Lyon, France), Professor P. Chambon (LGME-U184, Strasbourg, France), Dr. P. K. Vogt (The Scripps Institute, La Jolla, CA), Dr. P. B. Antin and Dr. C. P. Ordhal (San Francisco, CA), Dr. D. Montarras (Institut Pasteur, Paris, France), Dr. C. Deschene (Institut National de la Santé et de la Recherche Médicale, Nice, France), and Dr. M. Castellazzi (ENS Lyon) for the gift of pRSV cErbA{alpha}1 and pRSV CMD1, pSV Gal4{alpha}1 and Gal4-tk-CAT, BTG1, DR4-tk-CAT reporter construct, –73colCAT reporter gene and pRS chicken c-Jun expression plasmid, the myoblast QM7 line, pEMSV hMyf5 and pRSV Myogenin, c-Jun eb1, and c-Jun{Delta}176, respectively.


   Footnotes
 
This work was supported by the Institut National de Recherche Agronomique and grants from Association Française pour la recherche contre les Myopathies and Association pour la Recherche contre le Cancer.

Current address for L.D.: Laboratoire Biosciences de l’Aliment, Université Bordeaux I, ISTAB, USC-INRA 429, Avenue des Facultés, 33405 TALENCE Cedex, France.

Disclosure of potential conflicts of interest: M.B., L.D., P.S., S.G., L.P., F.C., C.W-C., and G.C. have nothing to declare.

First Published Online March 23, 2006

1 M.B. and L.D. have contributed an equal amount of work to this study. Back

Abbreviations: AP-1, Activator protein 1; bHLH, basic helix loop helix; CAT, chloramphenicol acetyltransferase; DR4, direct repeat 4; GST, glutathione-S-transferase; Ipal, inverted palindrome; pal, palindromic; RXR, retinoid X receptor; TR{alpha}1, T3 nuclear receptor {alpha}1; TRE, thyroid hormone response element.

Received January 27, 2006.

Accepted for publication March 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sugie H, Verity MA 1985 Postnatal histochemical fiber type differentiation in normal and hypothyroid rat soleus muscle. Muscle Nerve 8:654–660[CrossRef][Medline]
  2. King DM 1987 Thyroidal influence on nuclear accumulation and DNA replication in skeletal muscles of young chickens. J Expert Zool Suppl 1:291–298
  3. Whalen RG, Sell SM, Butler-Browne GS, Schwartz K, Bouveret P, Pinset- Haestrom I 1993 Three myosin heavy chain isozymes appear sequentially in rat muscle development. Nature 292:805–809
  4. Butler-Browne GS, Herlicoviez D, Whalen RG 1984 Effects of hypothyroidism on myosin isozyme transition in developing rat muscle. FEBS Lett 166:71–75[CrossRef][Medline]
  5. Izumo S, Nadal-Ginard B, Madhavi V 1986 All members of the MHC multigene family respond to thyroid hormone in highly tissue-specific manner. Science 231:597–600[Abstract/Free Full Text]
  6. Hoffman RK, Lazar MA, Rubinstein MA, Kelly AM 1994 Differential expression of {alpha}1, {alpha}2 and ß1 thyroid hormone receptor genes in developing rat skeletal muscle. J Cell Biochem 18D(Suppl):517
  7. Yu F, Gothe S, Wikstrom L, Forrest D, Vennstrom B, Larsson L 2000 Effects of thyroid hormone receptor gene disruption on myosin isoform expression in mouse skeletal muscles. Am J Physiol Regul Integr Comp Physiol 278:545–554
  8. Marchal S, Cassar-Malek I, Pons F, Wrutniak C, Cabello G 1993 Triiodothyronine influences quail myoblast proliferation and differentiation. Biol Cell 78:191–197[CrossRef][Medline]
  9. Carnac G, Albagli-Curiel O, Vandromme M, Pinset C, Montarras D, Laudet V, Bonnieu A 1992 3,5,3'-Triiodothyronine positively regulates both MyoD1 gene transcription and terminal differentiation in C2 myoblasts. Mol Endocrinol 6:1185–1194[Abstract/Free Full Text]
  10. Daury L, Busson M, Tourkine N, Casas F, Cassar-Malek I, Wrutniak-Cabello C, Castellazzi M, Cabello G 2001 Opposing functions of ATF2 and Fos-like transcription factors in c-Jun-mediated myogenin expression and terminal differentiation of avian myoblasts. Oncogene 20:7998–8008[CrossRef][Medline]
  11. Cassar-Malek I, Marchal S, Rochard P, Casas F, Wrutniak C, Samarut J, Cabello G 1996 Induction of c-Erb A-AP-1 interactions and c-Erb A transcriptional activity in myoblasts by RXR. Consequences for muscle differentiation. J Biol Chem 271:11392–11399[Abstract/Free Full Text]
  12. Downes M, Mynett-Johnson L, Muscat GE 1994 The retinoic acid and retinoid X receptors are differentially expressed during myoblast differentiation. Endocrinology 134:2658–2661[Abstract/Free Full Text]
  13. Daury L, Busson M, Casas F, Cassar-Malek I, Wrutniak-Cabello C, Cabello G 2001 The triiodothyronine nuclear receptor c-ErbA{alpha}1 inhibits avian MyoD transcriptional activity in myoblasts. FEBS Lett 508:236–240[CrossRef][Medline]
  14. Antin PB, Ordahl CP 1991 Isolation and characterization of an avian myogenic cell line. Dev Biol 143:111–121[CrossRef][Medline]
  15. Samuels HH, Stanley F, Casanova J 1979 Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105:80–85[Abstract/Free Full Text]
  16. Malik S, Huang CF, Schmidt J 1995 The role of the CANNTG promoter element (E box) and the myocyte-enhancer-binding-factor-2 (MEF-2) site in the transcriptional regulation of the chick myogenin gene. Eur J Biochem 230:88–96[Medline]
  17. Angel P, Baumann I, Stein B, Delius H, Rahmsdorf HJ, Herrlich P 1987 12-O-Tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region. Mol Cell Biol 7:2256–2266[Abstract/Free Full Text]
  18. Forman BM, Yang CR, Au M, Casanova J, Ghysdael J, Samuels HH 1989 A domain containing leucine-zipper-like motifs mediate novel in vivo interactions between the thyroid hormone and retinoic acid receptors. Mol Endocrinol 3:1610–1626[Abstract/Free Full Text]
  19. Lin ZY, Dechesne CA, Eldridge J, Paterson BM 1989 An avian muscle factor related to MyoD1 activates muscle-specific promoters in nonmuscle cells of different germ-layer origin and in BrdU-treated myoblasts. Genes Dev 3:986–996[Abstract/Free Full Text]
  20. Rowe A, Eager NS, Brickell PM 1991 A member of the RXR nuclear receptor family is expressed in neural-crest-derived cells of the developing chick peripheral nervous system. Development 111:771–778[Abstract]
  21. Vial E, Perez S, Castellazzi M 2000 Transcriptional control of SPARC by v-Jun and other members of the AP1 family of transcription factors. Oncogene 19:5020–5029[Medline]
  22. Castellazzi M, Loiseau L, Piu F, Sergeant A 1993 Chimeric c-Jun containing an heterologous homodimerization domain transforms primary chick embryo fibroblasts. Oncogene 8:1149–1160[Medline]
  23. Casas F, Rochard P, Rodier A, Cassar-Malek I, Marchal-Victorion S, Wiesner RJ, Cabello G, Wrutniak C 1999 A variant form of the nuclear triiodothyronine receptor c-ErbA{alpha}1 plays a direct role in regulation of mitochondrial RNA synthesis. Mol Cell Biol 19:7913–7924[Abstract/Free Full Text]
  24. Nielsen DA, Chou J, MacKrell AJ, Casadaban MJ, Steiner DF 1983 Expression of a preproinsulin-ß-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci USA 80:5198–51202[Abstract/Free Full Text]
  25. Wrutniak C, Cassar-Malek I, Marchal S, Rascle A, Heusser S, Keller JM, Flechon J, Dauca M, Samarut J, Ghysdael J, Cabello G 1995 A 43-kDa protein related to c-Erb A{alpha}1 is located in the mitochondrial matrix of rat liver. J Biol Chem 270:16347–16354[Abstract/Free Full Text]
  26. Casas F, Domenjoud L, Rochard P, Hatier R, Rodier A, Daury L, Bianchi A, Kremarik-Bouillaud P, Becuwe P, Keller J, Schohn H, Wrutniak-Cabello C, Cabello G, Dauça M 2000 A 45 kDa protein related to PPAR{gamma}2, induced by peroxisome proliferators, is located in the mitochondrial matrix. FEBS Lett 478:4–8[CrossRef][Medline]
  27. Busson M, Carazo A, Seyer P, Grandemange S, Casas F, Pessemesse L, Rouault JP, Wrutniak-Cabello C, Cabello G 2005 Coactivation of nuclear receptors and myogenic factors induces the major BTG1 influence on muscle differentiation. Oncogene 24:1698–1710[CrossRef][Medline]
  28. Snedecor GW 1961 Statistical methods. Ames, IA: Iowa State University Press; 534
  29. Bengal E, Ransone L, Scharfmann R, Dwarki VJ, Tapscott SJ, Weintraub H, Verma IM 1992 Functional antagonism between c-Jun and MyoD proteins: a direct physical association. Cell 68:507–519[CrossRef][Medline]
  30. Sugawara A, Yen PM, Darling DS, Chin WW 1993 Characterization and tissue expression of multiple triiodothyronine receptor-auxiliary proteins and their relationship to the retinoid X-receptors. Endocrinology 133:965–971[Abstract/Free Full Text]
  31. Andersson ML, Nordstrom K, Demczuk S, Harbers M, Vennstrom B 1992 Thyroid hormone alters the DNA binding properties of chicken thyroid hormone receptors {alpha} and ß. Nucleic Acids Res 20:4803–4810[Abstract/Free Full Text]
  32. Yen PM, Sugawara A, Chin WW 1992 Triiodothyronine (T3) differentially affects T3-receptor/retinoic acid receptor and T3-receptor/retinoid X receptor heterodimer binding to DNA. J Biol Chem 267:23248–23252[Abstract/Free Full Text]
  33. Ribeiro RC, Kushner PJ, Apriletti JW, West BL, Baxter JD 1992 Thyroid hormone alters in vitro DNA binding of monomers and dimers of thyroid hormone receptors. Mol Endocrinol 6:1142–1152[Abstract/Free Full Text]
  34. Perlmann T, Rangarajan PN, Umesono K, Evans RM 1993 Determinants for selective RAR and TR recognition of direct repeat HREs. Genes Dev 7:1411–1422[Abstract/Free Full Text]
  35. Kurokawa R, Yu VC, Naar A, Kyakumoto S, Han Z, Silverman S, Rosenfeld MG, Glass CK 1993 Differential orientations of the DNA-binding domain and carboxy-terminal dimerization interface regulate binding site selection by nuclear receptor heterodimers. Genes Dev 7:1423–1435[Abstract/Free Full Text]
  36. Hsu JH, Zavacki AM, Harney JW, Brent GA 1995 Retinoid-X receptor (RXR) differentially augments thyroid hormone response in cell lines as a function of the response element and endogenous RXR content. Endocrinology 136:421–430[Abstract]
  37. Marchal S, Cassar-Malek I, Magaud JP, Rouault JP, Wrutniak C, Cabello G 1995 Stimulation of avian myoblast differentiation by triiodothyronine: possible involvement of the cAMP pathway. Exp Cell Res 220:1–10[CrossRef][Medline]
  38. Desbois C, Aubert D, Legrand C, Pain B, Samarut J 1991 A novel mechanism of action for v-ErbA: abrogation of the inactivation of transcription factor AP-1 by retinoic acid and thyroid hormone receptors. Cell 67:731–740[CrossRef][Medline]
  39. Zhang XK, Hoffmann B, Tran PB, Graupner G, Pfahl M 1992 Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature 355:441–446[CrossRef][Medline]
  40. Lopez G, Schaufele F, Webb P, Holloway JM, Baxter JD, Kushner PJ 1993 Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site. Mol Cell Biol 13:3042–3049[Abstract/Free Full Text]
  41. Froeschle A, Alric S, Kitzmann M, Carnac G, Aurade F, Rochette-Egly C, Bonnieu A 1998 Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis. Oncogene 16:3369–3378[CrossRef][Medline]



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