A ras-dependent pathway abolishes activity of a muscle-specific enhancer upstream from the muscle creatine kinase gene.

Abstract

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 ...

Expression of the MyoD1 muscle determination gene defines differentiation capability but not tumorigenicity of human rhabdomyosarcomas.

“…This finding was similar to our previous results with an H-ras-transfected myogenic derivative from the C3H1OT1/2 cell line, where even in the presence of six copies of an actively transcribed H-ras gene, the muscle phenotype was maintained and could be enhanced by 5-azacytidine treatment (18). These results suggest that increased MyoDI expression may be able to overcome a ras-mediated block to the induction of musclespecific genes (34). A recent report that a TrpE-MyoDl fusion protein specifically binds to the mck 5' enhancer (J.…”

“…Other studies have approached the question of the relationship between differentiation and transformation in muscle cells by the introduction of transforming ras genes into myoblast cell lines (18,28,34). These experiments have shown that activated N-ras or H-ras genes can prevent skeletal myoblast differentiation in some (28) but not all (18) mouse cell lines.…”

“…These experiments have shown that activated N-ras or H-ras genes can prevent skeletal myoblast differentiation in some (28) but not all (18) mouse cell lines. Recently, Stemnberg et al (34) demonstrated that activated N-ras or H-ras expression abolished the activity of the muscle specific mck upstream enhancer and suggested that ras blocked the establishment of the myogenic phenotype by preventing the accumulation of regulatory factors required for transcriptional induction of muscle-specific genes. Here we have taken the opposite but complementary approach of introducing a determination gene into a human rhabdomyosarcoma cell line known to contain an activated N-ras gene (17).…”

A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes.

“…We and others have reported previously that the mck enhancer is contained within the region between bp -1350 and -1048 5' of the mck transcription initiation site (14,24,27,52,53). As a first step toward identification of nuclear factors that interact with the mck enhancer region, we divided the 302-bp enhancer into two parts by digestion with AvaI and analyzed factor interactions within each part by using gel retardation assays.…”

Distinct Effects of Rac1 on Differentiation of Primary Avian Myoblasts

“…Reporter plasmids for muscle-specific transcription included the pMCK-CAT plasmid (Sternberg et al, 1988), the 4R-tkCAT reporter (Weintraub et al, 1990), and the myogenin reporter construct pMyo1565CAT (kindly provided by E.N. Olson, University of Texas Southwestern Medical Center, Dallas, TX).…”