The transcription factor MyoD can stimulate myogenic conversion in a wide variety of cell types, for example in 10T1/2 fibroblasts, and is considered a master regulatory gene for myogenesis. It is unclear, however, why myogenic conversion is specific, since other transcription factors, for example the Drosophila neurogenic activator achaete-scute, bind to muscle-specific enhancers but fail to activate transcription. Similarly, sequence-specific binding of MyoD to the immunoglobulin enhancer does not stimulate transcription. The underlying hypothesis of this proposal is that the specificity of myogenic conversion depends upon a cofactor that recognizes elements of the basic region of myogenic activators, such as MyoD, as well as of the protein binding region of muscle-specific genes. The long-term objectives of this proposal are to identify such cofactors. The experimental design is based on the observations that in rhabdomyosarcoma (RD) cells MyoD does not activate muscle-specific promoters, and that in monkey kidney-derived CV-1 cells these promoters are activated by a mutated MyoD that is inactive in most other cells. Specifically, whole cell and microcell fusion between 10T1/2 and either RD or CV-1 cells will be used to study the importance of positive and negative regulators for transcriptional activation of a muscle-specific enhancer by MyoD and to determine how many chromosomes are involved in this process. Putative cofactors essential for the selective activation of muscle-specific genes by MyoD will then be cloned in a genetic screen. Alternatively, these factors will be isolated based on either their ability to accelerate in vitro transcription from a muscle-specific promoter in the presence of MyoD or their ability to associate with MyoD and DNA. The latter experiments will involve immunoprecipitation, crosslinking, or affinity chromatography on MyoD- binding site-containing columns. Overall, these studies will contribute to our understanding of muscle cell differentiation and will yield insight into the defects that result in lack of myogenic conversion and concomitant myosarcoma formation.
