Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, Università di Modena e Reggio Emilia, Via G. Campi 287, 41100 Modena, Italy
*Author for correspondence (e-mail: ferrari.stefano{at}unimo.it)
Accepted September 10, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Transcription factors, Cell compartmentation, Muscle differentiation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MEF2 activity is regulated by a variety of effectors in different cell types. Growth factor signalling mediated by mitogen-activated protein kinases (MAPKs), such as p38 and BMK1/ERK5, stimulates MEF2s transcriptional activity, by targeting phosphorylation to conserved sites in their TADs (Han et al., 1997; Kato et al., 1997). Calcium/calmodulin-dependent protein kinase (CaMK) and calcineurin also stimulate MEF2 activity (Woronicz et al., 1995; Liu et al., 1997, Wu et al., 2000). Five proteins have been identified so far that act as MEF2-specific transcriptional corepressors: Cabin 1 (Youn et al., 1999), MEF2 interacting transcriptional repressor (MITR) (Sparrow et al., 1999; Zhang et al., 2001) and the histone deacetylases HDAC4 (Miska et al., 1999), HDAC5 (Lemercier et al., 2000) and HDAC7 (Dressel et al., 2001). In particular, HDAC4, HDAC5 and HDAC7 are members of the class II histone deacetylases, which specifically interact with individual members of the MEF2 family through an N-terminal dimerization domain and repress MEF2 transcriptional activator capacity at various promoters (Miska et al., 1999; Lemercier et al., 2000; Dressel et al., 2001). Insight into the regulation and physiological significance of HDAC/MEF2 interactions has been provided by recent observations in different cell systems. In cardiomyocytes, activated CAMKIV disrupts the MEF2/HDAC complex and causes the unmasking of MEF2 transcriptional activity (Jianrong et al., 2000). This finding indicates that in cardiomyocytes MEF2 may be the target of hypertrophic signals that act through the CAMK pathway, or the MAPK pathway, or both (as in the case of signalling triggered by phenylephrine). In fact, while CaMK activates MEF2 by dissociating HDAC/MEF2 complexes, MAPK stimulates MEF2 activity by direct phosphorylation of the TAD. Together, the CAMK and MAPK pathways synergize to activate MEF2. Export of HDAC5 to the cytoplasm, following phosphorylation of specific serine residues by activated CAMKI, was shown to be essential for efficient myogenic conversion of 10T1/2 fibroblasts by exogenous MyoD (McKinsey et al., 2000), an event that requires MEF2 (Molkentin et al., 1995) and histone acetylase (HAT) activity (Sartorelli et al., 1997). In T cells, MEF2D was shown to play a key role in T-cell receptor (TCR)-mediated apoptosis during thymic negative selection. It mediates calcium-dependent transcription of Nur77, a transcription factor involved in TCR-mediated apoptosis of thymocytes (Woronicz et al., 1994; Liu et al., 1994). MEF2D was shown to bind two calcium-responsive DNA elements in the Nur77 promoter and to mediate the calcium-dependent induction of Nur77 (Woronicz et al., 1995). It was recently demonstrated that HDAC4 (as well as MITR) contains a calmodulin binding domain that overlaps with the MEF2 binding domain. Calcium-dependent binding of calmodulin to HDAC4 leads to its dissociation from MEF2, relieving MEF2 from the transcriptional repression of the Nur77 promoter by HDAC4 (Youn et al., 2000).
In the present report we provide a thorough study of the signals that mediate nuclear localization of MEF2 and show that the NLS present in MEF2 proteins is necessary to localize the HDAC4/MEF2 complex to the nucleus.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell cultures and transfections
C2.7 myoblasts (Pinset et al., 1988), kindly provided by M. Buckingham, were grown in Dulbeccos modified eagle medium (D-MEM, Life Technologies) containing 10% fetal calf serum (FCS, Hyclone). Differentiation to myotubes was achieved by lowering FCS concentration to 1%. Proliferating myoblasts, grown at 60% confluence in 60 mm dishes, were transfected with a total of 2 µg plasmid DNA/dish, using the lipid-based Lipofectamine Plus Reagent (Life Technologies) according to the manufacturers instructions.
Detection of GFP fluorescence and immunofluorescence
36-48 hours after transfection cells were washed extensively with PBS, fixed for 20 minutes at room temperature with 3% paraformaldehyde in PBS, permeabilized with 0.05% Triton X-100 in PBS for 5 minutes, and incubated for 15 minutes with PBS containing 1% bovine serum albumin (BSA). Cells were then incubated overnight at 4°C with the primary antibody at the appropriate dilution. Antibodies used were: monoclonal anti-Myc (1:100, Sigma), polyclonal anti-NFY-B (1:50) (Mantovani et al., 1992), polyclonal anti-HA (1:50, Sigma). Cells were then washed with PBS, incubated for 15 minutes with 1% BSA in PBS and incubated for further 60 minutes with the secondary antibody. Secondary antibodies used were: goat anti-mouse IgG rhodamine conjugated (1:200, Pierce), goat anti-rabbit IgG fluorescein-conjugated (1:200, Pierce). After extensive washing with PBS, cell monolayers were mounted in 10 mM Tris-HCl, pH 9, containing 60% glycerol and examined in a Zeiss Axiophot fluorescence microscope. Images were acquired to a digital camera by the SPOT32 software package (Diagnostic Instruments Inc.) and exported into Adobe PhotoShop for processing. Prints were obtained by employing a dye-sublimation printer (Kodak). Quantitative estimates of nuclear/cytoplasmic distribution of GFP/MEF2 proteins and HDAC4 were obtained by analyzing at least 100 cells in three different experiments. Green fluorescence intensity was scored by image analysis with Adobe PhotoShop and the obtained data were statistically evaluated with SPSS 9.0 software package.
Western blot analysis and immunoprecipitation
Proliferating C2.7 myoblasts in 100 mm dishes were transfected with 5 µg of pcHDAC4-Myc and 5 µg of pCMV-FLAG vector, pFLAG/MEF2C or pFLAG/MEF2CNLS. A fraction of the transfection cocktail was used to transfect C2.7 myoblasts grown in 30 mm dishes: these cells were subsequently analyzed by immunofluorescence to check the efficiency of transfection. 36 hours after transfection cells were collected and homogenized in 10 volumes of ice-cold buffer (10 mM Tris-HCl pH 7.9, 10 mM NaCl, 5 mM MgCl2, supplemented with protease inhibitors). KCl was then added to 100 mM final concentration, cytoplasmic protein extracts were harvested and nuclei were collected by centrifugation at 800 g. Nuclei were then homogenized in 100 µl of ice-cold 10 mM Tris-HCl pH 7.9, 5 mM MgCl2, 0.3 M KCl; nuclear extracts were harvested by centrifugation at 13,400 g for 20 minutes. 5 µg of cytoplamic and nuclear proteins were run in an SDS/polyacrylamide gel and transferred to a poly(vinylidene difluoride) membrane (Amersham). Filters were blocked by incubation with PBST (PBS containing 0.1% Tween-20), added with 5% low fat dry milk for 1 hour and then incubated with the primary antibodies in PBST overnight at 4°C. Antibodies used were: monoclonal anti-c-Myc antibody (1:1000, Sigma) or anti-flag M2 monoclonal antibody (1:1000, Sigma). Filters were extensively washed in PBST and then incubated with horseradish peroxidase conjugated secondary anti-mouse antibody (Amersham). After further extensive washes with PBST, antigen-antibody complexes were visualized with the enhanced chemiluminescence kit from Amersham. For the immunoprecipitation experiment, 50 µg of proteins were incubated with 50 µl of anti-flag M2 affinity gel (Sigma) in 500 µl of NET-gel buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, 0.25% gelatin and 0.02% Na-azide) supplemented with protease inhibitors, for 2 hours at 4°C. The affinity gel was washed twice with NET gel buffer and once with 10 mM Tris-HCl pH 7.5 and 0.1% NP-40. The affinity gel was resuspended in 60 µl of Laemmli buffer and boiled for 5 minutes; 6 µl were subjected to western blot analysis as described above.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this report we confirm that a C-terminal region encompassing aa 472-507 is necessary and sufficient for fully localizing GFP/MEF2A in the nucleus of C2C7 myogenic cells. Deletion of the C-terminal 36 amino acid sequence causes GFP/MEF2A to be completely retained in the cytoplasm. Indeed, as it had been pointed out previously (Yu, 1996), this region matches the bipartite NLS as originally proposed by Dingwall and Laskey (Dingwall and Laskey, 1991). Quite differently, the deletion of the corresponding region in MEF2C still allows a considerable proportion of the same protein to be retained in the nucleus. This result is surprising since the sequence homology of the C-terminal 36 amino acids in MEF2A and MEF2C is nearly complete. Exclusion of GFP/MEF2C from the nucleus is achieved when a region encompassing aa. 399-411 is deleted, thus suggesting that this sequence might represent an additional element specifically contributing to the nuclear localization or, more likely, to the nuclear retention of MEF2C. In fact, the analysis of the isolated C-terminal region of MEF2A and MEF2C as to the ability of targeting GFP to the nucleus, has shown that the C-terminal 36 amino acids of both proteins are equally effective as NLS. However, when an extension of further 30 amino acids of MEF2C is added to the bipartite NLS, GFP localization is exclusively nuclear. In particular, we hypothesize that the amino acid sequence 399-411 might represent or contain a signal for nuclear retention, rather than localization: in fact, when it is directly tethered to GFP, in the absence of the bipartite NLS, it does not enhance nuclear targeting. Sequence analysis of the C-terminal portion of MEF2C has shown that peptide 399-411 almost completely overlaps to a region of poor sequence homology to MEF2A (the sequence underlined in Fig. 2D, corresponding to aa. 397-409). The reason for such peculiarity is not known, as well as the mechanisms underlying the functional activity of the peptide: at present we can only hypothesize that it might function as a binding domain for some not yet identified nuclear component, ultimately causing MEF2C to be retained in the nucleus.
In addition we show that both MEF2A and MEF2C provide an NLS in trans for the selective nuclear import of HDAC4. Class II histone deacetylases (such as HDAC4, HDAC5 and HDAC7) have the ability of specifically interacting with MEF2 proteins, through an N-terminal dimerization domain (Miska et al., 1999; Lemercier et al., 2000; Dressel et al., 2001). However they show a very different subcellular distribution when transfected in C2.7 myogenic cells: in proliferating myoblasts HDAC4 is largely cytoplasmic or pancellular, while HDAC5 localizes almost exclusively in the nucleus. Upon differentiation to myotubes, HDAC5 moves to the cytoplasm, while HDAC4 does not significantly change its pattern of subcellular distribution. This observation is in agreement with recently published data obtained in Cos cells (McKinsey et al., 2000b) and suggests that the two deacetylases might rely on different mechanisms as far as their nuclear localization and, consequently, functional effect are concerned. As far as HDAC7 is concerned, a recent report shows that its subcellular distribution profile is essentially the same as HDAC5: nuclear in proliferating myoblasts and cytoplamic in growth arrested cells (Dressel et al., 2001). Co-transfection of either MEF2A or MEF2C with HDAC4 causes the deacetylase to switch to a predominant nuclear localization, mostly in the form of nuclear speckles, where MEF2 proteins are also detected. We show that this effect heavily depends on the NLS contributed by the MEF2 proteins as well as on the formation of a MEF2/HDAC4 heterodimer: in fact co-transfection of MEF2 NLS deletion mutants causes HDAC4 to enhance its cytoplasmic localization, while co-transfection of the MEF2 C-terminal region, which contains the NLS but no dimerization domain, does not have any effect on HDAC4 subcellular distribution. Therefore, the overall picture emerging from our observations is that, in proliferating myoblasts, HDAC5 (and possibly also HDAC7) has the intrinsic property of localizing to the nucleus, while HDAC4 requires the MEF2 NLS for efficiently translocating to the nucleus. Recently reported examples of proteins requiring heterodimerization and a trans-acting NLS for efficient localization to the nucleus are represented by the pituitary tumor-transforming gene product (PTTG) (Chien and Pei, 2000) and by the clock proteins mPER1 and mPER2 (Yagita et al., 2000). Both HDAC4 and HDAC5 have been shown to efficiently inhibit MEF2 transcriptional activity (Miska et al., 1999; Lemercier et al., 2000) and myogenesis (Lu et al., 2000). We also observed that, in C2.7 myoblasts, co-transfection of HDAC4 and MEF2A causes a tenfold reduction of the transcriptional activity at a MEF2 dependent promoter, compared with myoblasts transfected with MEF2A alone (data not shown). In this context an important role is played by 14-3-3 proteins and CaMK signalling. 14-3-3 proteins were shown to bind both HDAC4 and HDAC5 through interaction between specific domains (Wang et al., 2000; Grozinger and Schreiber, 2000) and negatively regulate their activity, apparently by sequestration in the cytoplasm or enhancement of nuclear export. However the two deacetylases are regulated differently by 14-3-3. In fact, ectopic co-expression of 14-3-3 and HDAC5 demonstrates that this deacetylase binds 14-3-3 and localizes to the cytoplasm only in the presence of CaMK; by contrast, HDAC4 appears to be constitutively bound to 14-3-3 (i.e. irrespectively of CaMK signalling) and mostly localized to the cytoplasm (McKinsey et al., 2000b). Our data are consistent with the hypothesis that HDAC4 is mostly a cytoplasmic resident protein, until its binding with 14-3-3 is substituted by MEF2, which is capable of heterodimerizing with HDAC4 in the cytoplasm and provides the signal for nuclear localization. On the contrary, HDAC5 would be a mostly nuclear resident protein, not requiring the interaction with any NLS-containing partner for localization. As it was consistently reported, HDAC4 (as well as HDAC5) is an effective repressor of MEF2 transcriptional activity; then the intriguing question arises about the functional significance of MEF2 carrying to the nucleus its own repressor. One might speculate that MEF2 factors, at least in cells expressing functionally significant levels of HDAC4, are negatively regulated by default. Repression relief might be achieved by either of the following two, non-mutually exclusive mechanisms: dissociation of HDAC4/MEF2 complexes, as was shown to be the case in HDAC5/MEF2 complexes after activation of CaMK signalling (McKinsey et al., 2000b); and substitution of HDAC4 by a transcriptional co-activator such as p300, which was shown to compete with HDAC4 binding to MEF2D (Youn et al., 2000).
![]() |
ACKNOWLEDGMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Black, B. L. and Olson, E. N. (1998). Transcriptional control of muscle development by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14, 167-196.[Medline]
Chien, W. and Pei, L. (2000). A novel binding factor facilitates nuclear translocation and transcriptional activation function of the pituitary tumor-transforming gene product. J. Biol. Chem. 275, 19422-19427.
Clarke, N., Arenzana, N., Hai, T., Minden, A. and Prywes, R. (1998). Epidermal growth factor induction of the c-jun promoter by a Rac pathway. Mol. Cell. Biol. 18, 1065-1073.
Dingwall, C. and Laskey, R. A. (1991). Nuclear targeting sequences a consensus? Trends Biochem. Sci. 16, 478-481.[Medline]
Di Silvio, A., Imbriano, C. and Mantovani, R. (1999). Dissection of the NF-Y transcriptional activation potential. Nucleic Acids Res. 27, 2578-2584.
Dressel, U., Bailey, P. J., Wang, S.-C. M., Downes, M., Evans, R. and Muscat, G. E. O. (2001). A dynamic role for HDAC7 in MEF2-mediated muscle differentiation. J. Biol. Chem. 276, 17007-17013.
Grozinger, C. M. and Schreiber, S. L. (2000). Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3-3-dependent cellular localization. Proc. Natl. Acad. Sci. USA 97, 7835-7840.
Han, J., Jiang, Y, Li, Z., Kravchenko, V. V. and Ulevitch, R. J. (1997). Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 386, 296-299.[Medline]
Han, T. H. and Prywes, R. (1995). Regulatory role of MEF2D in serum induction of the c-jun promoter. Mol. Cell. Biol. 15, 2907-2915.[Abstract]
Jianrong, L., McKinsey, T. A., Nicol, R. L. and Olson, E. N. (2000). Signal-dependent activation of the MEF2 transcription factor by dissociation from histone deacetylases. Proc. Natl. Acad. Sci. USA 97, 4070-4075.
Kato, Y., Kravchenko, V. V., Tapping, R. I., Han, J., Ulevitch, R. J. and Lee, J. D. (1997). BMK1/ERK5 regulates serum induced early gene expression through transcription factor MEF2C. EMBO J. 16, 7054-7066.
Lemercier, C., Verdel, A., Galloo, B., Curtet, S., Brocard, M.-P. and Khochbin, S. (2000). MHDA1/HDAC5 histone deacetylase interacts with and represses MEF2A transcriptional activity. J. Biol. Chem. 275, 15594-15599.
Lin, Q., Schwartz, J., Bucana, C. and Olson, E. N. (1997). Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science 276, 1404-1407.
Lin, Q., Lu, J., Yanagisawa, H., Webb, R., Lyons, G. E., Richardson, J. A. and Olson, E. N. (1998). Requirement of the MADS box trasnscription factor MEF2C for vascular development. Development 125, 4565-4574.
Liu, S., Liu, P., Borras, A., Chatila, T. and Speck, S. H. (1997). Cyclosporin A-sensitive induction of the Epstein-Barr virus lytic switch is mediated via a novel pathway involving a MEF2 family member. EMBO J. 16, 143-153.
Liu, Z. G., Smith, S. W., McLaughlin, K. A., Schwartz, L. M. and Osborne, B. A. (1994). Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene Nur77. Nature 367, 281-284.[Medline]
Lu, J., McKinsey, T. A., Zhang, C.-L. and Olson, E. N. (2000). Regulation of skeletal myogenesis by association of the MEF2 transcription factor with class II histone deacetylases. Mol. Cell 6, 233-244.[Medline]
Mantovani, R., Pessara, U., Tronche, F., Li, X.-Y., Knapp, A.-M., Pasquali, J.-L., Benoist, C. and Mathis, D. (1992). Monoclonal antibodies to NF-Y define its function in MHC classII and albumin gene transcription. EMBO J. 11, 3315-3322.[Abstract]
Mao, Z., Bonni, A., Xia, F., Nadal-Vicens, M. and Greenberg, M. E. (1999). Neuronal activity-dependent cell survival mediated by transcription factor MEF2. Science 286, 785-790.
Martin, J. F., Schwartz, J. J. and Olson, E. N. (1993). Myocyte enhancer factor (MEF) 2C: a tissue-restricted member of the MEF-2 family of transcription factors. Proc. Natl. Acad. Sci. USA 90, 5282-5286.[Abstract]
McKinsey, T. A., Zhang, C.-L, Lu, J. and Olson, E. N. (2000a). Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408, 106-111.[Medline]
McKinsey, T. A., Zhang, C.-L. and Olson, E. N. (2000b). Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5. Proc. Natl. Acad. Sci. USA 97, 14400-14405.
Miska, E. A., Karlsson, C., Langley, E., Nielsen, S. J., Pines, J. and Kouzarides, T. (1999). HDAC4 deacetylase associates with and represses the MEF2 transcription factor. EMBO J. 18, 5099-5107.
Molkentin, J. D., Black, B. L., Martin, J. E. and Olson, E. N. (1995). Cooperative activation of muscle gene expression by MEF2and myogenic bHLH proteins. Cell 83, 1125-1136.[Medline]
Pinset, C., Montarras, D., Chenevert, J., Minty, A., Barton, P., Laurent, C. and Gros, F. (1988). Control of myogenesis in the mouse myogenic C2 cell line by medium composition and by insulin: characterization of permissive and inducible C2 myoblasts. Differentiation 38, 28-34.[Medline]
Olson, E. N., Perry, M. and Schultz, R. A. (1995). Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors. Dev. Biol. 172, 2-14.[Medline]
Sartorelli, V., Huang, J., Hamamori, Y. and Kedes, L. (1997). Molecular mechanisms of myogenic coactivation by p300: direct interaction with the activation domain of MyoD and with the MADS box of MEF2C. Mol. Cell. Biol. 17, 1010-1026.[Abstract]
Sparrow, D. B., Miska, E. A., Langley, E., Reynaud-Deonauth, S., Kotecha, S., Towers, N., Spohr, G., Kouzarides, T. and Mohun, T. J. (1999). MEF-2 function is modified by a novel co-repressor, MITR. EMBO J. 18, 5085-5098.
Verdel, A., Curtet, S., Brocard, M.-P., Rousseaux, S., Lemercier, C., Yoshida, M. and Khochbin, S. (2000). Active maintenance of mHDA2/mHDAC6 histone deacetylase in the cytoplasm. Curr. Biol. 10, 747-749.[Medline]
Wang, A. H., Kruhlak, M. J., Wu, J., Bertos, N. R., Vezmar, M., Posner, B. I., Bazett-Jones, D. P. and Yang, X.-J. (2000). Regulation of histone deacetylase 4 by binding of 14-3-3 proteins. Mol. Cell. Biol. 20, 6904-6912.
Woronicz, J. D., Calnan, B., Ngo, V. and Winoto, A. (1994). Requirement for the orphan receptor Nur77 in apoptosis of T-cell hybridomas. Nature 367, 277-281.[Medline]
Woronicz, J. D., Lina, A., Calnan, B. J., Szychowski, S., Cheng, L. and Winoto, A. (1995). Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis. Mol. Cell. Biol. 15, 6364-6376.[Abstract]
Wu, H., Naya, F. J., McKinsey, T. A., Mercer, B., Shelton, J. M., Chin, E. R., Simard, A. R., Michel, R. N., Bassel-Duby, R., Olson, E. N. and Williams, R. S. (2000). MEF2 responds to multiple calcium-regulated signals in the control of skeletal muscle fiber type. EMBO J. 19, 1963-1973.
Yagita, K., Yamaguchi, S., Tamanini, F., van der Horst, G. T. J., Hoeijmakers, J. H. J., Yasui, A., Loros, J. L., Dunlap, J. C. and Okamura, H. (2000). Dimerization and nuclear entry of mPER proteins in mammalian cells. Genes Dev. 14, 1353-1363.
Youn, H. D., Sun, L., Prywes, R. and Liu, J. O. (1999). Apoptosis of T cells mediated by Ca2+-induced release of the transcription factor MEF2. Science 286, 790-793.
Youn, H.-D., Grozinger, C. M. and Liu, J. O. (2000). Calcium regulates transcriptional repression of myocyte enhancer factor 2 by histone deacetylase 4. J. Biol. Chem. 275, 22563-22567.
Yu, Y.-T. (1996). Distinct domains of myocyte enhancer binding factor-2A determining nuclear localization and cell type-specific transcriptional activity. J. Biol. Chem. 271, 24674-24683.
Yu, Y.-T., Breitbart, R. E., Smoot, L. B., Lee, Y., Mahdavi, V. and Nadal-Ginard, B. (1992). Human myocyte-specific enhancer factor 2 comprises a group of tissue-restricted MADS box transcription factors. Genes Dev. 6, 1783-1798.[Abstract]
Zhang, C. L., McKinsey T. A. and Olson, E. N. (2001). The transcriptional corepressor MITR is a signal-responsive inhibitor of myogenesis. Proc. Natl. Acad. Sci. USA 98, 7354-7359.