Differentiation of skeletal muscle involves an ordered series of morphological and molecular events in which undifferentiated myoblasts exit the cell cycle and fuse to form multi-nucleated myotubes. During this period, an array of muscle-specific genes encoding proteins required for the specialized functions of the mature muscle fiber is induced to high levels. Myogenic differentiation is suppressed by fibroblast growth factor (FGF) and transforming growth factor type-beta (TGF-beta) and by mutationally activated ras proteins, which are postulated to couple growth factor receptors with intracellular effectors. The goals of this project are designed to define more precisely the nuclear targets for the cascades triggered by growth factors and ras proteins and to identify mechanism(s) involved in transcriptional induction of muscle-specific genes during myogenesis. To address this problem, we have isolated the mouse muscle creatine kinase (mck) gene, which is among the tissue-specific genes that are induced during myogenesis. We have also identified a distal upstream enhancer and a weaker intragenic regulatory element confer differentiation-specific and cell-type-restricted expression on this gene. A series of myoblast cell lines that express ras autonomously also has been constructed. The program for differentiation of these cells is subject to negative control by ras. In the present study we propose to fully characterize mck regulatory elements and to determine sites for interaction of specific DNA binding proteins with these elements. The ways in which growth factor and ras-dependent pathways impinge on these mck regulatory factors also will be examined. Finally, we will purify and eventually obtain cDNA clones for mck regulatory factors which will allow investigations into the cascade of regulatory events that controls establishment of the myogenic phenotype.
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