) The molecular basis for cessation of cell division and terminal differentiation of specialized tissues such as skeletal muscle is just beginning to be understood. The transcription factor MyoD is required for muscle determination but is expressed in cells prior to terminal differentiation. Rhabdomyosarcoma is a common childhood malignancy of skeletal muscle that expresses MyoD yet fails to terminally differentiate. Discoveries in the laboratory have shown a genetic lesion responsible for suppression of the MyoD dependent phenotype. This lesion is the amplification of the MDM2 locus from chromosome 12 translocated to chromosome 14. The goal of this application is to understand how MDM2 inhibits MyoD function. Preliminary results suggest that MDM2 inhibits MyoD indirectly by binding to another muscle-specific transcription factor MEF2C. The first specific aim of this application is to identify the precise domains of each protein required for this interaction and to construct mutant proteins that fail to interact. The techniques used include co-immunoprecipitation and the split hybrid system, a novel variant of the yeast two hybrid system designed to screen for disruption of a protein-protein interaction. The second specific aim of this application is to characterize the biologic activity of the mutants made in the first specific aim. Transfection of the mutant cDNAs and assays of muscle differentiation will be used test the hypothesis that interaction with MEF2C is required for MDM2 to inhibit myogenesis. The third specific aim is to precisely define the mechanism by which MDM2 inhibits myogenesis. The hypotheses that MDM2 disrupts MEF2C interactions with DNA and with MyoD will be tested. MDM2's effects on MyoD and MEF2C interactions with the transcription co-activator, CBP will also be examined. These studies will not only specifically clarify the molecular basis for oncogenesis in rhabdomyosarcoma but will aid the general understanding of cell-type determination. The basic concepts will likely be applicable to other terminally differentiated, non-dividing cells including neurons and cardiac myocytes. In this mentored career development plan, the Principal Investigator will enhance his knowledge of molecular and developmental biology through course work and advanced training in a supervised research setting. He will gain expertise in experimental design and an appreciation of the principles of responsible scientific conduct. At the end of the five-year mentorship period he will have achieved independence in the design and implementation of biomedical research.
