Although significant progress has been made in the molecular understanding of skeletal muscle differentiation, little is known concerning genes involved in the development of cardiac muscle. The applicant's long- term goal is to develop a novel expression cloning method or to utilize the newly developed """"""""interaction trap"""""""" method in order to identify such cardiac regulatory factors that may not be easily isolated by conventional methods. The first plan would be to develop a new method of expression cloning in mammalian cells in which expressed cDNAs could be isolated based on their ability to activate expression of a reporter gene linked to a cis-acting enhancer sequence. This method may identify factors that do not directly bind to DNA, but act as tissue-specific cofactors that interact with and activate other tissue specific or ubiquitous transcription factors. The second expression cloning strategy would be to utilize the interaction trap method in yeast. The applicant plans to isolate cDNA clones from proteins that interact with the MADS box-containing protein MEF-2 or the helix-loop-helix proteins E2-2 or Id), in leu-host. A cardiac cDNA library would be made in a vector that allows expression of cDNA encoded proteins fused to an acidic transcription activation domain (""""""""prey"""""""") in yeast. When a cDNA encoded """"""""prey"""""""" is able to associate with a """"""""bait"""""""" fusion protein, the Leu 2 reporter gene is activated, which renders the leu-host cells viable in a selection medium. The applicant would use a new and improved version of the interaction trap method developed by Roger Brent. Once novel clones are isolated, the applicants would determine the sequence and pattern of gene expression of these new factors. The applicant would also determine the mechanism of trans- activation of previously characterized cardiac muscle specific promoters by these factors using various assays, including co-transfection, DNA binding, co-immunoprecipitation and site-directed mutagenesis. In addition, in order to examine the role these factors may play in control of the cardiac myogenic program, the applicant would over-express the cloned factor in mouse embryonic stem cells that are capable of differentiating into cardiac myocytes in vitro. If time permits, the applicants would create ES cell lines that carry homozygous """"""""knock- out"""""""" of the factor genes and examine their capability to differentiate into cardiac myocytes in vitro.
