Oncogene (1998) 16, 3369 ± 3378  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis

Anne Froeschle 1,SeÂverine Alric1, Magali Kitzmann2, Gilles Carnac2, Fre de ric Aurade 3, Ce cile Rochette-Egly4 and Anne Bonnieu1

1Laboratoire de Di€eÂrenciation Cellulaire et Croissance Institut National de la Recherche Agronomique; Place Pierre Viala 34060 Montpellier Cedex 1; 2Cell Biology Unit, Centre de Recherches de Biochimie MacromoleÂculaire, CNRS, BP 5051, 34033 Montpellier Cedex; 3Institut Pasteur, DeÂpartement de Biologie MoleÂculaire, URA 1148 CNRS, 75724 Paris Cedex 15; 4Institut de GeÂneÂtique et de Biologie MoleÂculaire et Cellulaire, (GBMC) BP 163, 67404 Illkirch Cedex, CU de Strasbourg, France

The results reported here indicate that retinoic acid (RA) receptor superfamily (GigueÁ re, 1994; Glass, 1994). induces growth arrest and di€erentiation only in MyoD- These combinatorial interactions result in a ®ne tuning expressing muscle cells. Transient transfection assays of RA-receptor activity and could explain how RA reveal a functional interaction between MyoD, a key elicits highly pleiotropic e€ects on the di€erentiation of myogenic regulator and RA-receptors, principal media- numerous cell types. tors of RA actions. Interestingly, we demonstrate that Di€erentiation of skeletal muscle cells sequentially RXR-MyoD-containing complexes are recruited at requires growth arrest followed by the onset of a timely speci®c MyoD DNA-binding sites in muscle cells. ordered expression of muscle-speci®c genes. This Furthermore, we also demonstrate that RA-receptors programme depends on the myogenic regulatory and the muscle basic helix ± loop ± helix (b-HLH) proteins, including the basic helix ± loop ± helix (b- proteins interact physically. Mutational analysis suggests HLH) proteins MyoD, myogenin, Myf5, MRF4 that this interaction occurs via the basic region of muscle (Weintraub et al., 1991; Lassar and Munsterberg, b-HLH proteins and the DNA-binding domain of RA- 1994; Olson and Klein, 1994) and the MADS domain receptors and is important for functional interactions transcription factor MEF2 (Gossett et al., 1989; Yu et between these two families of transcription factors. In al., 1992; Olson et al., 1995). The muscle-speci®c b- conclusion, these results highlight novel interactions HLH factors dimerize with an ubiquitous class of b- between two distinct groups of regulatory proteins that HLH E-proteins for activation of muscle-speci®c genes in¯uence cell growth and di€erentiation. (Murre et al., 1989; Lassar et al., 1991; Hu et al., 1992). Furthermore, MyoD also associates with several Keywords: retinoic acid receptors; muscle b-HLH non-b-HLH proteins to control withdrawal from cell proteins; protein-protein interaction; myogenesis; cycle and induction of the myogenic phenotype (Bengal et al., 1992; Gu et al., 1993; Kaushal et al., 1994; Schneider et al., 1994; Eckner et al., 1996; Groisman et al., 1996; Molkentin et al., 1995; Puri et al., 1997). Introduction Compelling evidence in the literature indicates that RA induces myogenesis of various muscle cells (Gabbert et Retinoic acid (RA), a natural derivative of vitamin A, al., 1988; Arnold et al., 1992; MomoõÈ et al., 1992; regulates the growth and di€erentiation of a wide Albagli-Curiel et al., 1993; Halevy and Lerman, 1993). variety of cell types (De Luca, 1991; Chambon, 1994; In the case of C2 myoblasts, RA is an ecient inducer Gudas et al., 1994; Hofmann and Eichele, 1994). These of both growth arrest and di€erentiation (Albagli- actions are mediated by two families of ligand- Curiel et al., 1993). These e€ects have been associated inducible transcriptional factors, the retinoic acid with an RA-induction of MyoD and myogenin gene receptors (RARa, b and g) and the retinoid X expression. These observations suggest that RA- receptors (RXRa, b and g) which are members of the receptors and MyoD might act in the same regulatory nuclear receptor superfamily (Evans, 1988; Chambon, pathway that governs the RA-induced myogenesis. 1994; GigueÁ re, 1994; Gronemeyer and Laudet, 1995; Here, we investigated the functional and biochemical Mangelsdorf and Evans, 1995). RARs bind and are relationships between RA-receptors and MyoD that activated by all-trans RA and its 9-cis isomer (Zelent et in¯uence cell growth and di€erentiation in muscle cells. al., 1989; Allenby et al., 1993) whereas RXRs bind and Using various muscle cell lines which di€er in their are activated by 9-cis RA only (Heyman et al., 1992; pattern of MyoD expression, we found that the Levin et al., 1992; Mangelsdorf et al., 1992). RARs and promyogenic e€ect of RA is correlated with the RXRs transregulate target gene expression by binding presence of this muscle transcription factor. Consistent as heterodimers to speci®c RA response elements with these data we further show that RA-receptors and (RAREs). Furthermore, RXRs also serve as hetero- MyoD up-regulate each other's transcriptional activity. dimeric partners for other members of the nuclear We establish that this coactivation involves a direct association of RA-receptors with myogenic b-HLH factors and propose that the interaction of RA- receptors and myogenic b-HLH proteins is important Correspondence: A Bonnieu for the execution of RA-induced myogenic differentia- The ®rst two authors contributed equally to this work Received 28 October 1997; revised 2 February 1998; accepted 3 tion. The demonstration that the members of the February 1998 nuclear receptor superfamily (RAR and RXR) and the MyoD-RA receptors interactions in muscle cells Froeschle et al 3370 MyoD family can physically interact establishes a cell line stably transfected with a vector expressing mechanism for myogenic activation by retinoic acid. sense MyoD RNA (referred to as L6M for L6 MyoD) (this work). Control (C2C and L6C) and transfected (C2AM and L6M) cells were tested for their MyoD gene expression. As expected, C2C and L6M cells Results express MyoD whereas C2AM and L6C cells do not express MyoD as determined by immuno¯uorescence MyoD is required for mediating the RA signal in analysis (Figure 1a). We then examined these cell lines myogenesis for their sensitivity to RA with respect to their In C2 myoblasts, RA rapidly increases MyoD gene proliferation and di€erentiation properties. expression (Albagli-Curiel et al., 1993) suggesting a RA-induced myogenic di€erentiation in these cell possible role of this myogenic factor in the mediation lines was tested by immuno¯uorescence analysis. of RA-induced skeletal myogenesis. To test this Myoblasts were induced to di€erentiate in low serum possibility, we compared the e€ects of RA in mouse medium (Figure 1) supplemented with RA (1076 M) for C2 and rat L6 myogenic cell lines which di€er in 4 days, then immunostained for troponin T, a late MyoD gene expression (Braun et al., 1989; Wright et muscle marker. Cells expressing MyoD (C2C and al., 1989). Unlike C2 cells, L6 myoblasts do not express L6M) depicted an increased staining for troponin T MyoD. We also investigated the RA sensitivity of C2 protein in response to RA (Figure 1a). By contrast, cells in which MyoD gene expression was inhibited and cells in which MyoD is absent (C2AM and L6C) are of L6 cells overexpressing MyoD. For this purpose, we largely insensitive to RA-induced di€erentiation. The used a previously-described C2 cell line which C2AM cell line failed to di€erentiate when treated with constitutively produces antisense MyoD RNA (re- RA. Note that, unlike C2AM cells, L6C myoblasts ferred to as C2AM for C2 Anti-MyoD) (Montarras expressed the muscle-speci®c marker upon RA-treat- et al., 1996). Moreover, we established an L6 myogenic ment, but a similar di€erentiation was also obtained in

a C2C C2AM C RA C RA

MyoD

Troponin T

Hoechst

L6C L6M C RA C RA

MyoD

Troponin T

Hoechst MyoD-RA receptors interactions in muscle cells Froeschle et al 3371 a medium devoid of RA (Figure 1a, Table 1). acid receptors and the MyoD factor. Reporter Therefore, MyoD expression appeared to be required constructs containing a retinoic acid response element for myoblast di€erentiation in the presence of RA. (TREpalX3-tk-CAT or RAREb-tk-CAT) were transi- Changes in the RA responsiveness of these cell lines ently cotransfected with or without plasmids expressing were further investigated by measuring cell growth in an RA-receptor (RARa or RXRa) and MyoD in the presence and absence of RA. Cells were plated in a mouse C3H10T1/2 ®broblasts (Figure 2a and b). These proliferative medium (Figure 1) and treated or not with cells do not express MyoD but are `permissive' for its 1076 M RA, then proliferation was evaluated using functions (Davis et al., 1987). In presence of RA, [3H]thymidine incorporation experiments. As shown in TREpalX3-tk-CAT activity was increased upon RARa Figure 1b. RA strongly inhibited growth of myoblasts or RXRa overexpression. Strikingly, a stronger (two- expressing MyoD (C2C and L6M). This inhibitory and fourfold more) ligand-dependent reporter activity e€ect was detectable after 2 days and after 4 days was obtained when RARa or RXRa was cotransfected reached about 90% relative to the value in non-treated with MyoD. This stimulation of RA-induced CAT cells. By contrast, RA did not signi®cantly prevent activity is MyoD dose-dependent (Figure 2a). Analo- proliferation of the MyoD-defective cells (C2AM and gous results were obtained with the RAREb-tk-CAT L6C) (Figure 1b). Therefore, RA-induced growth reporter gene (Figure 2b). These results indicate that arrest is only observed in the myogenic cell lines MyoD potentiates the transactivation of RA-receptors expressing MyoD. In conclusion, these data establish and support a role for MyoD as a positive cofactor in that the RA-induced growth arrest and di€erentiation the retinoic acid receptor signalling pathway. in muscle cells are directly linked to the presence of Reciprocally, we investigated whether RA-receptors MyoD. could increase MyoD transcriptional activity. C3H10T1/2 cells were cotransfected with a reporter construct containing the E-box DNA-binding sites RA-receptors and MyoD can co-operate in transcriptional activation

Sense or antisense MyoD RNA expression could Table 1 Percentage of cells expressing Troponin T at day 4 of induce changes in the expression and function of di€erentiation nuclear RA-receptors (RARs and RXRs) which are the C2C C2AM L6C L6M major mediators of RA e€ects. Northern blots 7RA +RA 7RA +RA 7RA +RA 7RA +RA indicated that the pattern of RAR and RXR Troponin T 2 ± 5 20 ± 25 0 0 2 ± 5 3 ± 5 3 ± 6 20 ± 30 expression is independent of the presence or absence The percentages correspond to the ratio of the cells stained with an of MyoD in each of both L6 and C2 cell lines (data not anti-troponin T antibody by immuno¯uorescence analysis versus the shown). We then performed transfection experiments total number of nuclei stained with Hoechst. The percentages are to test for a functional interaction between retinoid determined after counting more than 103 cells for each point

b

Figure 1 RA-induced di€erentiation and growth arrest in C2 and L6 myoblasts are linked to MyoD gene expression. The control C2 cells (C2C) and C2 anti-MyoD cells (C2AM) have previously been described (Montarras et al., 1996). L6 cells (clone L6G7) (Ya€e and Saxel, 1977) were cotransfected with 10 mg of pEMSV vectors containing or lacking the murine MyoD coding sequence (respectively designated L6C and L6M) and 500 ng of pSV2neo. The transfected L6 cells were selected for 2 weeks in proliferation medium (Dulbecco's modi®ed Eagle's medium (DMEM) (1 vol) / Ham F-12 (1 vol) supplemented with 10% Fetal Calf Serum (FCS)) with G418 (1 mg/ml). Individual colonies were isolated, then passaged into stable cell lines. (a) C2C, C2AM, L6C and L6M 76 cells were induced to di€erentiate in DMEM with 2% FCS and all-trans RA (10 M) for 4 days, then double-immunostained with troponin T monoclonal antibody (Sigma) and MyoD antiserum (Vandromme et al., 1995) as indicated. In all cases, nuclei were stained with Hoechst B2883 dye. (b) These four cell lines were maintained in proliferation medium and treated daily for 4 days with 76 3 10 M all-trans RA. Their proliferative capacity in presence of RA was evaluated by [ H]thymidine incorporation. For each cell line, the proliferation index is expressed each day as a percentage of the ratio of [3H]thymidine incorporation of RA-treated cells versus the non-treated ones. The proliferative capacities of L6 and C2 control cell lines (L6C and C2C) are arbitrary, taken as 100% the ®rst day of RA-treatment. Plotted values represent the average obtained from triplicate dishes (+s.d.) MyoD-RA receptors interactions in muscle cells Froeschle et al 3372

Figure 2 Functional interaction between RA-receptors and MyoD. (a) Mouse C3H10T1/2 ®broblasts were transiently transfected as described in Materials and methods with 1 mg of TREpalX3-tk-CAT reporter construct, 0.25 mg of RA-receptor expression vector (pSG5-RXRa or pSG5-RARa) and pEMSV-MyoD expression vector (at increasing concentrations of 0.25 and 2 mg) as indicated. The plotted values represent the average of seven independent experiments. Standard deviations between the experiments are less than 10% (s.d.510%). `None' indicates transfection experiments performed without MyoD, RARa and RXRa; `7', transfection experiments performed without MyoD. (b) The reporter construct RAREb-tk-CAT (1 mg) was tested in parallel with cotransfected pEMSV-MyoD expression vector (at increasing concentrations of 0.25 and 2 mg) as indicated. The plotted values represent the average of three independent experiments (s.d.510%). `None' indicates transfection experiments performed without MyoD. (c) 1 mg of TkCAT reporter gene construct containing E-box DNA-binding site from AchRa promoter was transiently cotransfected in C3H10T1/2 cells with 0.25 mg of pEMSV-MyoD expression plasmid and RA-receptor expression vector (pSG5-RARa or pSG5- RXRa) (at increasing concentrations of 0.25 and 2 mg) as indicated. 24 h after transfection, the cells were incubated in the absence 76 (7) or presence (+) of 10 M all-trans RA. The plotted values represent the average of three independent experiments (s.d.510%). `None' indicates transfection experiments performed without MyoD, RARa and RXRa; `7', transfection experiments performed without RARa or RXRa. CAT activities are expressed relative to those in untreated TREpalX3-tk-CAT, RAREb-tk- CAT or E-box-tk-CAT pSG5/pEMSV-transfected control cells (arbitrarily taken as 1)

from the a- subunit promoter of mouse acetylcholine display E-box DNA-binding activities (two complexes receptor (E-box-tkCAT), the expression vectors encod- marked myo/E, Figure 3, lanes 1 and 7, solid arrows). ing MyoD and an RA-receptor (RARa or RXRa). In The binding speci®city of these complexes was the absence of RA, MyoD overexpression activated the con®rmed by competition experiments with excess of AchR-a subunit promoter (Olson and Klein, 1994; and unlabelled E-box-speci®c oligonucleotide (lanes 2 and Figure 2b). In the presence of RA, this basal CAT 8). These complexes were speci®cally supershifted by a transactivity was enhanced about twofold suggesting a MyoD-speci®c antibody (lanes 4 and 10), and most functional implication of endogenous RA-receptors in likely represent heterodimeric complexes between C3H10T1/2 cells. A maximal RA-e€ect on MyoD myogenic and ubiquitous b-HLH proteins (Weintraub activity was obtained when MyoD was coexpressed et al., 1991). Note that the MyoD antibody preferen- with one of the two RA-receptors (RARa or RXRa). tially supershifts the high (*) and to a lesser extent the Thus, MyoD appeared to be more ecient (about 2 ± 4- low (**) molecular weight myo/E complex, most likely fold) in activating the E-box reporter gene in the because its epitope was not exposed. In addition, two presence of ligand-activated RA-receptors (Figure 2b). di€erent monoclonal antibodies directed against either This RA-potentiation of MyoD activity was dependent RXR (a, b and g) or RXRa both supershifted these on RA-receptor concentrations (Figure 2b) and complexes (preferentially the low mobility myo/E required intact E-box DNA-binding sites in the complex **) (lanes 5, 6, 11 and 12), demonstrating AchR-a subunit promoter (data not shown). These the presence of RXRa in E-box DNA-binding activity. data show that MyoD potentiates retinoid acid The speci®city of this supershift was con®rmed using receptor activity in the induction of RA-responsive either an unrelated monoclonal antibody (lanes 3 and elements and reciprocally that RA-receptors stimulate 9) or by adding the antibodies to the probe directly in the activation capacity of MyoD in the presence of RA. the absence of C2 cell extracts, which does not result in any supershifted band (data not shown). Similar results were observed using an oligonucleotide bearing two E- RA-receptors are present in the protein complex formed boxes (data not shown). From these results, we on the speci®c MyoD DNA target sequences conclude that RXRa constitutes part of the speci®c To investigate whether RA-receptors and myogenic b- E-box-bound complexes. HLH proteins can be detected in a common protein complex, we performed electrophoretic mobility shift Interaction between RA-receptors and myogenic b-HLH assay (EMSA) experiments using a 32P-labelled single proteins occurs through DNA-binding domains E-box oligomer speci®c for MyoD DNA binding (Bengal et al., 1994). Nuclear extracts of undiffer- To test whether or not RA-receptors can directly entiated C2 myoblasts and di€erentiated C2 myotubes interact with MyoD, we then performed in vitro MyoD-RA receptors interactions in muscle cells Froeschle et al 3373 γ

γ of b-HLH proteins (Figure 4b). Among the MyoD , , β β , , mutants tested, internal deletion mutants encompassing D D α α α α o o R R R R

. . the basic domain (amino acids 102 ± 114) and the basic- y y X X X X p p e e M R R M R R

®rst helix junction (amino acids 102 ± 135), failed to n n m m b b b b b b s o s o o o n A A A n c n A A A n c bind GST- RXRa. Similar results were obtained using RXRs GST-RARa resin (data not shown). Thus, the basic + myo/E domain of muscle b-HLH factors is necessary for myo/E * binding RA-receptors. Furthermore, these MyoD ** mutants which did not interact with RA-receptors failed to potentiate the transcriptional activity of RA- receptors (as measured by the TREpalX3-tk-CAT in C3H10T12 cell line) (Figure 5a). To further analyse this association, RXRa, RXRb and various deletion mutants as well as other members of the steroid receptor superfamily (THRb thyroid hormone recep- torb and ERa: estrogen receptor a) were radiolabelled in vitro and allowed to react with the GST-MyoD fusion protein (Figure 4c). Interestingly, RXRa, RXRb 1 2 3 4 5 6 7 8 9 10 11 12 and THRb, but not ERa bound GST-MyoD, Myoblasts Myotubes suggesting that MyoD recognition could be restricted Figure 3 RXRa localizes at E-box MyoD DNA-binding sites. to a subset of nuclear hormonal receptors (see Electrophoretic mobility shift assay using nuclear-cell extracts discussion). Among the RA-receptor mutants tested, from C2 myoblasts (lanes 1 ± 6) and from di€erentiated C2 only mutant RA-receptors containing an intact DNA- myotubes (cultured for 48 h in low serum medium) (lanes 7 ± 12). binding domain eciently interact with GST-MyoD A gel shift probe containing one E-box was employed. Lanes 1 (Figure 4c). Similar results were obtained with RARa and 7 show the gel shift pattern obtained with extract alone. In lanes 2 and 8, a 100-fold excess of cold E-box oligonucleotide was (data not shown). This suggests that RA-receptor- added to the gel shift reaction. For the other lanes, the antibodies MyoD interaction requires the DNA-binding domain indicated at the top were added to the binding reaction. A speci®c of RA-receptors. Accordingly, the mutant GST- supershift was obtained with antibodies directed against the b- RXRaDABC which has the N-terminal A/B and the HLH MyoD protein (lanes 4 and 10), as well as with the 4RX- 1D12 (directed against RXRa, b and g) and 4RX3A2 (directed DBD regions deleted failed to interact with MyoD against RXRa) monoclonal RXR antibodies (lanes 5, 11 and 6, (Figure 4d). As a control, this mutant fusion protein 12 respectively) but not with a speci®c non-cross-reactive was still capable of interacting with RARa (Figure 4d). monoclonal antibody directed against CDK2 (noted ns: lanes 3 As reported in Figure 5b, the DBD-deletion RA- and 9). Complexes marked myo/E correspond to E-box DNA- receptor which did not interact with MyoD failed to binding activities. Two RXR antibodies (lanes 5, 6 and 11, 12) supershifted these complexes (marked RXRs + MyoD). Open potentiate the MyoD transcriptional activity (measured arrows indicate complexes supershifted with MyoD antibody by the E-box-tkCAT in C3H10T1/2 cell line). In summary (Figure 5c), our results establish that RA- receptors and the muscle b-HLH proteins physically glutathione-S-transferase (GST) pull-down experi- interact through DNA-binding domains. In addition, ments. We ®rst examined the ability of GST fusion the ability of RA-receptors and MyoD to upregulate protein containing full-length MyoD (GST-MyoD) to each other's transcriptional activity correlates well with speci®cally bind in vitro-translated full-length RARa their abilities to interact physically, suggesting that this and RXRa (Figure 4a, right panel). In both the interaction is important for functional interactions presence or absence of RA, 35S-methionin-labelled between these two families of transcription factors. RXRa and RARa bound recombinant GST-MyoD with high eciency, but did not interact with the control GST protein. In the reverse experiments, Discussion RARa or RXRa-GST resins (GST-RXRa or GST- RARa) also retained speci®cally in vitro-translated The data reported here present new evidence for MyoD (Figure 4a left panel). These results con®rm functional cooperation between the two RA-receptor that the two families of RA-receptors physically families and MyoD in the mediation of anti-prolif- interact with MyoD transcriptional factor in an RA- erative and di€erentiation e€ects of RA in muscle cells. independent manner. This interaction was not due to We found that the promyogenic e€ect of RA di€ers in residual DNA since it was also observed in the various muscle cell lines and is correlated with the presence of ethidium bromide, which prevents DNA- presence of MyoD. Consistent with these data, we dependent protein-protein interaction (data not further show that RA-receptors and MyoD upregulate shown). each other's transcriptional activity in the presence of The domains of each polypeptide required for this RA: this cooperation requires physical interaction interaction were mapped by similar in vitro protein between myogenic factors and RA-receptors. binding experiments (Figure 4b, c and d). E12, myogenin, MyoD b-HLH proteins and various MyoD RA biological activities are dependent on MyoD gene deletion mutants were translated in vitro and analysed expression for association with the GST-RXRa fusion protein (Figure 4b). Interestingly, MyoD and myogenin, but RA is known to cause a reduction of cell growth not E12, eciently bound GST-RXRa indicating that combined with di€erentiation of many cell types (De RXRa interacts speci®cally with the myogenic subclass Luca, 1991; Chambon, 1994; Gudas et al., 1994; MyoD-RA receptors interactions in muscle cells Froeschle et al 3374 Hofmann and Eichele, 1994). In cultured myoblasts, al., 1990; Sorrentino et al., 1990). Part of the mechanism this RA-antiproliferative e€ect is necessary but not by which MyoD and RA initiate and maintain the sucient to trigger myogenesis (Froeschle et al., 1996). growth arrest stage of di€erentiation might be explained Interestingly, MyoD also inhibits cell cycle progression by direct interaction of these two factors with several independently of myogenic di€erentiation (Crescenzi et cell cycle regulatory proteins (Bengal et al., 1992; D D

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GST-MyoD Figure 4 In vitro mapping of domains required for RA-receptors and muscle b-HLH protein interaction. (a) RA-receptors and MyoD interact in vitro. GST pull-down using in vitro-translated and labelled MyoD or RA-receptors (either RXRa, RARa) (10% of input shown) incubated with, respectively, GST-RA receptors (GST-RXRa or GSR-RARa) (left panel) and GST-MyoD (right 76 panel) beads in the absence (7) or presence (+) of 10 M all-trans RA. The GST controls exhibit no speci®c binding. (b) The basic domain of MyoD is necessary for binding RA-receptors. Similar experiment to (a) with in vitro-translated and labelled E12, myogenin, MyoD and various deletion mutants of MyoD incubated with GST-RXRa resin. The various in vitro-translated 35S-Met- labelled MyoD proteins (2 ml of programmed reticulocyte lysates per lane) were subjected to SDS-PAGE (left panel). Equal amounts of in vitro translation products were used in the binding assays (right panel). (c and d) DNA-binding domain of RA- receptors is required for speci®c MyoD recognition. (c) Similar experiments to (a) with in vitro-translated and labelled ERa THRb, RXRa, RXRaDAB, RXRaDEF, RXRb, RXRbDC incubated with GST-MyoD resin. The various in vitro-translated 35S-Met- labelled nuclear receptors (2 ml of programmed reticulocyte lysates per lane) were subjected to SDS-PAGE (left panel). Equal amounts of in vitro translation products were used in the binding assays (right panel). (d) GST pull-down using in vitro-translated and labelled MyoD incubated with GST-RXRa and GST-RXRaDABC resins. As a positive control, radiolabelled RARa was incubated with GST-RXRaDABC resin MyoD-RA receptors interactions in muscle cells Froeschle et al 3375

Figure 5 Functional interaction between RA-receptors and MyoD correlates with their ability to interact physically. Transient transfection protocol was as described in Figure 2. C3H10T1/2 cells were cotransfected with the reporter plasmid indicated above each panel and expression plasmids encoding the proteins listed below each bar. Plotted values represent the average of three separately performed experiments. The variation between the experiments is less than 10% (s.d.510%). (a) Transfection of a mixture of 0.25 mg of RA-receptor expression vector (pSG5-RXRa or pSG5-RARa) and 2 mg of pEMSV-MyoD expression plasmid shows the maximal activation e€ect of RA-receptors by MyoD (see Figure 2a). For comparison, the same amounts (mg) in the ratio of RA-receptors/MyoD mutants 1 : 8 were also transfected. (b) Transfection of a mixture of 0.25 mg of pEMSV-MyoD expression plasmid and 2 mg of RA-receptor expression vector (pSG5-RXRa or pSG5-RARa) shows the maximal activation e€ect of MyoD by activated RA-receptors (see Figure 2b). 2b). For comparison, the same amounts (mg) in the ratio of MyoD/RA-receptor mutants 1 : 8 were also transfected. (c) Summary of results in Figures 4 and 5. A `+' indicates binding; `7', no binding. C/H, cysteine- histidine-rich region; b, basic domain; HLH, helix-loop-helix region; C, DNA-binding domain (DBD), LBD, ligand- binding domain. ND: not determined

Schneider et al., 1994; Groisman et al., 1996). Here, we that RA induces myogenin expression in a rhabdo- have shown that ligand-activated RA-receptors require myosarcoma cell line, probably by regulating MyoD the presence of MyoD to prevent proliferation in C2 activity (Arnold et al., 1992). Here, we report that RA- and L6 myoblasts. This observation indicates that these receptors activate the endogenous myogenic pro- two families of transcription factors cooperate in gramme only in MyoD-positive cells. This e€ect can mediating the RA-antiproliferative e€ect which is a be partly explained by the RA-potentiation of MyoD prerequisite for muscle terminal di€erentiation. activity, and reciprocally, as demonstrated in transient We previously observed that in C2 myoblasts, RA is transfection experiments. Thus, a functional interaction able to enhance MyoD and myogenin gene expression between RA-receptors and myogenic factors might be (Albagli-Curiel et al., 1993) and thereby probably involved in both induction and maintenance of the improves the autoregulation and interregulation loops myogenic phenotype. of myogenic b-HLH factors, leading to increased amounts of muscle transcription factors that are Functional cooperation between RA-receptors and MyoD required for activation of the myogenic programme factors requires direct protein-protein interactions (Thayer et al., 1989). It is also possible that, besides enhancing expression of myogenic b-HLH factors, RA The cooperation between RA-receptors and myogenic might also regulate their activity. This is strongly b-HLH proteins to mediate RA-responsive events in suggested by our previous ®nding that RA abrogates muscle cells suggests that these factors interact the transactivating function of the transcription factor physically. Here, we report that these two regulatory AP1 (Albagli-Curiel et al., 1993), an inhibitor of both protein families interact through the DNA-binding myogenic factor expression and activity (Bengal et al., domain of RA-receptors and the basic domain of 1992; Li et al., 1992). In addition, it has been shown muscle factors comprising the myogenic function. We MyoD-RA receptors interactions in muscle cells Froeschle et al 3376 further show that MyoD recognition is restricted to RA transcriptional-activation signals in muscle cells. class II of nuclear receptors as MyoD binds to RAR, Further studies are necessary to determine precisely RXR and THR, but not ER (a member of class I of how the RA-receptor/myogenic b-HLH factor interac- nuclear receptors). Interestingly, RA-receptors do not tion stimulates the basal transcriptional machinery. In interact with the ubiquitous E12 b-HLH protein. Thus, conclusion, association between RA-receptors and the muscle speci®city of this interaction results from myogenic b-HLH factors could explain the coordina- the discrimination by RA-receptors between myogenic tion and cross-coupling of RA-induced growth arrest and non-myogenic b-HLH domains. Moreover, this and myogenic di€erentiation. It is thus possible that speci®c interaction could underlie the RA e€ects on speci®c actions of RA-receptors in a wide variety of muscle cells. Indeed, we show that functional coopera- cell types may derive from their interaction with cell- tion between RA-receptors and myogenic b-HLH speci®c regulatory molecules. proteins correlates with their abilities to interact with each other, suggesting the importance of the physical interaction between these two transcription factor Materials and methods families. It is also possible that other factors mediate Cell culture products functional collaboration. The members of the myo- Dulbecco's modi®ed Eagle's medium (DMEM), nutrient cyte-speci®c enhancer factor 2 (MEF2) family could be mixture F-12 (Ham) and Fetal Calf Serum (FCS) were candidate molecules for such co-regulatory activities. purchased from Gibco-BRL. All-trans retinoic acid (RA) MEF2 participates in the induction and maintenance was obtained from Sigma. All-trans RA was diluted in of muscle-di€erentiated phenotype by inducing the dimethyl sulfoxide (DMSO). expression of, and directly associating with, the myogenic b-HLH proteins (Kaushal et al., 1994; Cell culture conditions Molkentin and Olson, 1996). MEF2 has also been C3H10T1/2 ®broblasts were grown in Dulbecco's modi®ed shown to interact with members of the nuclear receptor Eagle's medium (DMEM) supplemented with 10% Fetal family, however it binds weakly to RAR and not at all Calf Serum (FCS) (Rezniko€ et al., 1973). C2 (clone C2.7) to RXR (Lee et al., 1997). Taken together with the and L6 (clone L6G7) myoblasts have been previously ®nding that the degree of functional cooperation described (Ya€e and Saxel, 1977). All cultures were correlated well with that of protein-protein interac- performed at 378C in a humidi®ed atmosphere of air with 5% CO . Proliferating myoblasts were routinely main- tion, we propose that direct interactions between RA- 2 receptors and MyoD are crucial for the mediation of tained in growth medium (GM), a 1 : 1 mixture of Ham F- 12/DMEM supplemented with 10% (vol/vol) Fetal Calf RA e€ects in muscle cells. Serum (FCS) and subcultured twice a week. Before any treatment with all-trans RA, cells were grown for about 7 Molecular mechanisms of myogenic coactivation by days in GM containing 10% hormone-depleted FCS. RA-receptors Depleted serum was obtained using the resin procedure of Samuels et al. (1979). All RA treatments were performed Given that unliganded receptors are already involved at about 48 h after plating at a density of 26103 cells/cm2. in this direct protein-protein association and are part For di€erentiation, cells plated at a density of 46103 cells/ of the MyoD protein complex on the E-box, the cm2 were grown for 3 days in GM containing 10% speci®c action of RA might lie in its ability to stimulate hormone-depleted FCS then transferred into differentia- the transcriptional activity of the complex RA- tion medium consisting of DMEM and 2% hormone- depleted FCS. The RA treatment was administered when receptors/myogenic factors. Comparison of structures the medium was changed. Control cells received solvent of unliganded with liganded RA-receptors (Bourguet et alone (0.1% DMSO). al., 1995; Renaud et al., 1995; Wurtz et al., 1996) suggests that RA-binding induces a conformational change allowing the receptors to interact with several Reporter expression vectors and other plasmids putative transcriptional intermediary factors required The reporter constructs TREpalx3-tk-CAT, RAREb-tk- for RA-dependent transcriptional activity (Le Douarin CAT have been previously described (Froeschle et al., et al., 1995; Schulman et al., 1995; Swaeld et al., 1996). The construct E-box-tk-CAT is referred to as 1995; vom Baur et al., 1995; May et al., 1996). Thus, paAChCAT+ (Piette et al., 1990). through these positive cofactors/mediators, only the GST-RARa, GST-RXRa and GST-RXRaDABC, pre- viously referred to as GSTRXRa(DE), in pGEM vector liganded-complex RA-receptors/myogenic factors were described (Le Douarin et al., 1995; Schulman et al., might act eciently on the basal transcription 1995; Swaeld et al., 1995; vom Baur et al., 1995; May et al., machinery, as recently described for complexes 1996). pSG5-RARa, pSG5-RXRa, pSG5-RXRaDAB and involving nuclear receptors (Fondell et al., 1993; Lee pSG5-RXRaDEF (previously referred to as pSG5-RXRadn) et al., 1994; Valcarcel et al., 1994; Chakravarti et al., expression vectors have been described (Durand et al., 1992; 1996). Consistent with this view, p300/CBP have been Nagpal et al., 1992). The RXRbDC expression vector, pSG5- reported acting as coactivators for RA-receptors but RXRbDC contains a RXRbcDNA lacking the DBD domain, also for the transcriptional factor MyoD (Chakravarti referred to as pcxn2RXRbDBD7 (Minucci et al., 1994), et al., 1996; Eckner et al., 1996; Yuan et al., 1996; Puri which was cloned into pSG5 and controlled by sequence. et al., 1997; Sartorelli et al., 1997). Because p300/CBP pEMSV-MyoD was a kind gift of Dr H. Weintraub (Davis et al., 1987). MyoD mutants previously referred to as DM have histone acetyl transferase activity and thereby can 63 ± 99, DM 102 ± 114, DM 102 ± 135, in pEMSV vector lack contribute directly to transcriptional regulation respectively the C/H domain, the basic domain, the basic- (Ogryzko et al., 1996), it is conceivable that the helix1 region (Davis and Weintraub, 1992). Additional interaction of p300/CBP with the complex RA- proteins included RXRb (Zelent et al., 1989), THRb receptors/MyoD may be important for integrating (Weinberger et al., 1986), ERa (Green et al., 1986), MyoD-RA receptors interactions in muscle cells Froeschle et al 3377 myogenin (Schwarz et al., 1992), E12 (Trouche et al., 1993) 30 min before the DNA-binding reaction was started. The and GST-MyoD (Groisman et al., 1996). following antibodies were used: 5.8A MyoD monoclonal antibody (Dako), 4RX-1D12 (directed against RXRa, b and g) and 4RX3A2 (directed against RXRa) monoclonal DNA transfections and CAT assays RXR antibodies (Rochette-Egly et al., 1994) and M22 Transfections and CAT assays were performed as described Cdk2 polyclonal antibody (Tebu) used as a control. (Froeschle et al., 1996), except that C3H10T1/2 cells were transfected. Transfections of plasmid DNA were performed In vitro transcription-translation and GST-binding assays using DOTAP reagent (Boehringer) as described by the supplier. Brie¯y, cells were transfected with 1 mg of One microgram of various supercoiled DNA plasmids was reporter constructs, 2 mg of b-galactosidase expression transcribed in vitro and then translated in the presence of vector (PCH110-Pharmacia) and 2.25 mg of a mixture of [35S]methionine with the TnT-coupled reticulocyte lysate vectors expressing MyoD, RARa or RXRa (Figures 2 and system (Promega) according to the manufacturer's sugges- 5). Cells were exposed to the DNA for 8 h then refed with tions. DMEM/HamF-12 medium supplemented with 10% de- Expression and puri®cation of glutathione S-transferase pleted FCS. RA treatment (1076 M) was performed 24 h (GST) fusion proteins were performed as described by Gu et after transfection. al. (1993). Determination of CAT activity was performed as To perform a protein-protein interaction assay, GST previously described (Pfahl et al., 1990). The b-galactosidase fusion proteins coupled to glutathione-agarose beads activity was measured as previously described (Nilsen et al., (Pharmacia) were incubated with various in vitro-translated 1983) to normalize for transfection eciency. proteins overnight at 48C in binding bu€er (20 mM HEPES

(pH 7.9), 50 mM KCl, 2.5 mM MgCl2 10% glycerol, 1 mM DTT). Then the glutathione beads were collected by brief DNA-binding assays centrifugation and washed ®ve times with RIPA bu€er Nuclear cell extracts of exponentially growing or differ- (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, entiated C2 cells were prepared as follows. Cells were 0.2% Nonidet P-40 detergent, 1 mM PMSF). For each washed with ice-cold phosphate-bu€ered saline (PBS) and reaction tube, equal amounts of GST fusion proteins or in resuspended in lysis bu€er (10 mM Tris pH 7.5, 10 mM vitro-translated 35S-Met-labelled proteins were used. The

NaCl, 3 mM MgCl2, 0.5% NP40 detergent, 4 mM dithio- protein amounts were estimated to the band intensities by threitol (DTT), 10 mg of leupeptin per ml, 10 mg of Coomassie blue staining or autoradiography (Radioactivity aprotinin per ml). The cells were centrifugated at 48C, on the gels was determined on a PhosphorImager analyzer). and the pellet was resuspended in nuclear bu€er (30 mM Bound proteins were boiled in sodium dodecyl sulfate (SDS) Tris pH 7.9, 350 mM NaCl, 5 mM DTT, 10% glycerol, loading bu€er, resolved by SDS-polyacrylamide gel electro- 20 mM sodium ¯uoride, 20 mM glycophosphate, 10 mg of phoresis (PAGE), and detected by autoradiography. leupeptin per ml, 10 mg of aprotinin per ml). After 30 min incubation on ice, cell nuclear extracts were clari®ed by Acknowledgements centrifugation at 13 000 r.p.m. Nuclear extracts were We thank Drs H Weintraub, EN Olson, P Chambon, K combined in 20 ml of binding reaction bu€er containing Ozato for the various plasmids used in this work. We are

12.5 mM Tris pH 7.9, 50 mM KCl, 5 mM MgCl2, 7.5% also indebted to Drs P Vigneron, F Bacou, V Cavailles, D glycerol, 0.1 mM DTT, 0.5 mg of poly(dI-dC) and 32P-end- Trouche, S Leibovitch, C Pinset, D Montarras, O Albagli- labelled E-box probe (5'-GATCCAGCAGGTGTGAAT- Curiel, C Prades and D Piquemal for fruitful discussions TCG-3'). After 15 min at room temperature, samples were and critical reading of the manuscript. This work was subjected to electrophoresis in a 4% non-denaturing supported by grants from the Association FrancË aise contre polyacrylamide gel. For competition experiments, 100-fold les Myopathies (AFM), the Association pour la Recherche molar excess of unlabelled E-box oligonucleotide was sur le Cancer (ARC), and the Institut National de la added to the reaction mixture prior to addition of Recherche Agronomique (INRA). AF and SA are the extract. For antibody shift EMSAs, concentrated anti- recipients of a doctoral fellowship from, respectively, the bodies were added to extracts at room temperature for ARC and the Ligue contre le Cancer.

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