Genetic, molecular and computational analysis of MEF2 function in Drosophila myogenesis. The goal of this proposal is to define the molecular mechanisms by which the transcription factor Myocyte enhancer factor-2 (MEF2) activates target gene expression. Several studies have identified critical roles for MEF2 in formation of the musculature and in differentiation of other tissues including immune cells and neurons. Moreover, variants in MEF2 orthologs in humans are associated with cardiac disease and autism. However despite the importance of MEF2 to muscle formation and human disease, a relatively small number of co-factors have been identified that function alongside MEF2 to participate in myogenesis, and no systematic or genome-wide approaches have been identified to understand how MEF2 controls gene expression. Moreover, it is not clear how MEF2 interacts with the basal transcription machinery. In this proposal, we will use the power of the Drosophila system, that has a single Mef2 gene, to execute a three- pronged approach to identify and characterize factors that interact with MEF2. In Aim 1, we will continue and expand a genetic modifier screen to identify genes for which haploinsufficiency enhances a Mef2 mutant phenotype. In preliminary data we demonstrate the feasibility of this approach, and identify a number of potential co-factors to be characterized. In Aim 2, we will carry out a molecular screen to identify factors that co-immune purify with MEF2 from embryonic lysates. In preliminary data we demonstrate that MEF2 interacts with CF2, that was identified in our earlier studies of MEF2 co-factors. In Aim 3, we will continue a bioinformatic analysis of MEF2 target genes, to identify sequences that are enriched in MEF2 target enhancers and promoters, and to identify and characterize the factors that interact with these sequences. In preliminary data we demonstrate the feasibility of this approach by showing that our independent bioinformatic analyses identify sites for two known MEF2 co-factors, and we identify additional binding activities that might also represent MEF2 co-factors. In Aim 4, we will collate the factors identified in Aims 1-3 and select for mechanistic analysis those that have high probability to be MEF2 co-factors. Overall our proposed experiments, which are all based upon strong preliminary data, will provide new insight into mechanisms by which MEF2 functions in animals. Given the strong conservation in the sequence and function of MEF2 within the animal kingdom, our findings will have direct impact upon our understanding of MEF2 function in mammalian development and disease.