Exposure of skeletal myoblasts to growth factor-deficient medium results in transcriptional activation of muscle-specific genes, including the muscle creatine kinase gene (mck). Tissue specificity, developmental regulation, and high-level expression of mck are conferred primarily by a muscle-specific enhancer located between base pairs (bp) -1350 and -1048 relative to the transcription initiation site (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909. To begin to define the regulatory mechanisms that mediate the selective activation of the mck enhancer in differentiating muscle cells, we have further delimited the boundaries of this enhancer and analyzed its interactions with nuclear factors from a variety of myogenic and nonmyogenic cell types. Deletion mutagenesis showed that the region between 1,204 and 1,095 bp upstream of mck functions as a weak muscle-specffic enhancer that is dependent on an adjacent enhancer element for strong activity. This adjacent activating element does not exhibit enhancer activity in single copy but acts as a strong enhancer when multimerized. Gel retardation assays combined with DNase I footprinting and diethyl pyrocarbonate interference showed that a nuclear factor from differentiated C2 myotubes and BC3H1 myocytes recognized a conserved A+T-rich sequence within the peripheral activating region. This myocyte-specific enhancer-binding factor, designated MEF-2, was undetectable in nuclear extracts from C2 or BC3H1 myoblasts or several nonmyogenic cell lines. MEF-2 was first detectable within 2 h after exposure of myoblasts to mitogen-deficient medium and increased in abundance for 24 to 48 h thereafter. The appearance of MEF-2 required ongoing protein synthesis and was prevented by fibroblast growth factor and type transforming growth factor, which block the induction of muscle-specific genes. A myoblast-specific factor that is down regulated within 4 h after removal of growth factors was also found to bind to the MEF-2 recognition site. A 10-bp sequence, which was shown by DNase I footprinting and diethyl pyrocarbonate interference to interact directly with MEF-2, was identified within the rat and human mck enhancers, the rat myosin light-chain (m1c)-113 enhancer, and the chicken cardiac mkc-2A promoter. Oligomers corresponding to the region of the mlc-113 enhancer, which encompasses this conserved sequence, bound MEF-2 and competed for its binding to the mck enhancer. These results thus provide evidence for a novel myocyte-specific enhancer-binding factor, MEF-2, that is expressed early in the differentiation program and is suppressed by specific polypeptide growth factors. The ability of MEF-2 to recognize conserved activating elements associated with multiple muscle-specific genes suggests that this factor may participate in the coordinate regulation of genes during myogenesis.Development requires precisely coordinated control of transcription. Differentiation of skeletal myoblasts to myotubes represents a well-defined sy...

Abstract. Type 13 transforming growth factor (TGFI3) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation. The effects of TGFI3 also are cell-type specific and appear to be modulated by other growth factors. In the present study, we examined the potential of TGFI3 for control of myogenic differentiation. In mouse C-2 myoblasts, TGFfl inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor. Differentiation of the nonfusing muscle cell line, BC3H1, was also inhibited by TGFI3 in a dose-dependent manner (IDs0 ~0.5 ng/ml). TGFI3 was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation. Inhibition of differentiation required the continual presence of TGFI3 in the culture media. Removal of TGFI3 led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGFfl are highly transient and require continuous occupancy of the TGFI~ receptor. Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGFI3 inhibited differentiation at the level of muscle-specific mRNA accumulation. These results provide the first demonstration that TGFI3 is a potent regulator of myogenic differentiation and suggest that TGFI3 may play an important role in the control of tissue-specific gene expression during development.

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Exposure of skeletal myoblasts to growth factor-deficient medium results in transcriptional activation of muscle-specific genes, including the muscle creatine kinase gene (mck). Tissue specificity, developmental regulation, and high-level expression of mck are conferred primarily by a muscle-specific enhancer located between base pairs (bp) -1350 and -1048 relative to the transcription initiation site (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909, 1988). To begin to define the regulatory mechanisms that mediate the selective activation of the mck enhancer in differentiating muscle cells, we have further delimited the boundaries of this enhancer and analyzed its interactions with nuclear factors from a variety of myogenic and nonmyogenic cell types. Deletion mutagenesis showed that the region between 1,204 and 1,095 bp upstream of mck functions as a weak muscle-specific enhancer that is dependent on an adjacent enhancer element for strong activity. This adjacent activating element does not exhibit enhancer activity in single copy but acts as a strong enhancer when multimerized. Gel retardation assays combined with DNase I footprinting and diethyl pyrocarbonate interference showed that a nuclear factor from differentiated C2 myotubes and BC3H1 myocytes recognized a conserved A + T-rich sequence within the peripheral activating region. This myocyte-specific enhancer-binding factor, designated MEF-2, was undetectable in nuclear extracts from C2 or BC3H1 myoblasts or several nonmyogenic cell lines. MEF-2 was first detectable within 2 h after exposure of myoblasts to mitogen-deficient medium and increased in abundance for 24 to 48 h thereafter. The appearance of MEF-2 required ongoing protein synthesis and was prevented by fibroblast growth factor and type beta transforming growth factor, which block the induction of muscle-specific genes. A myoblast-specific factor that is down regulated within 4 h after removal of growth factors was also found to bind to the MEF-2 recognition site. A 10-bp sequence, which was shown by DNase I footprinting and diethyl pyrocarbonate interference to interact directly with MEF-2, was identified within the rat and human mck enhancers, the rat myosin light-chain (mlc)-1/3 enhancer, and the chicken cardiac mlc-2A promoter. Oligomers corresponding to the region of the mlc-1/3 enhancer, which encompasses this conserved sequence, bound MEF-2 and competed for its binding to the mck enhancer. These results thus provide evidence for a novel myocyte-specific enhancer-binding factor, MEF-2, that is expressed early in the differentiation program and is suppressed by specific polypeptide growth factors. The ability of MEF-2 to recognize conserved activating elements associated with multiple-specific genes suggests that this factor may participate in the coordinate regulation of genes during myogenesis.

Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909. To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes.During skeletal-muscle differentiation, myoblasts cease dividing and express a series of more than 20 unlinked tissue-specific genes (9,15,17,46). The pattern of expression of these genes is most easily explained in terms of shared regulatory factors that confer muscle specificity through interaction with cis-acting regulatory elements associated with these genes. Indeed, evidence for the involvement of positive-and negative-regulatory factors in the control of muscle-specific genes has been obtained in transfection studies involving the use of muscle-regulatory elements linked to reporter genes (3, 6, 29, 30, 32-34, 42-44, 62) and in heterokaryon experiments in which myotubes are fused to nonmyogenic cells (4,5,66,67). It has also been demonstrated in C2 myoblasts that trans-acting factors required for induction of the skeletal and cardiac ox-actin genes may accumulate prior to differentiation (42, 57), suggesting that additional regulatory events such as changes in chromatin configurations (10, 13), DNA methylation (69), modification of factors, or synthesis of accessory proteins may influence the expression of the differentiated phenotype.Serum components and several peptide growth factors have been demonstrated to suppress myogenic differentiation through a mechanism independent of ...

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific genes whose products are required for the specialized functions of the mature muscle fiber. The program for myogenic differentiation is subject to negative control by several peptide growth factors and by the products of mutationally activated ras oncogenes, which persistently activate intracellular cascades normally triggered by specific growth factors. Previously, we reported that induction of the muscle creatine kinase (mck) gene during myogenesis was dependent on a distal upstream enhancer that cooperated with a proximal promoter to direct high levels of expression in developing muscle cells (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, T. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909). To investigate the mechanisms whereby ras blocks the induction of muscle-specific genes, we have examined the ability of mck 5' regulatory elements to direct expression of the linked reporter gene for chloramphenicol acetyltransferase (cat) in C2 myoblasts bearing mutant N-ras and H-ras oncogenes. In this paper we report that expression of activated ras alleles abolishes activity of the mck upstream enhancer but does not affect the activity of the mck promoter. The ability of ras to repress the expression of mck-cat fusion genes that have been transfected either transiently or stably into myoblasts suggests that ras may exert its effects on muscle-specific genes through mechanisms independent of chromatin configurations or DNA methylation. These results also suggest that ras blocks establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes.