The primary goal of this proposal is to characterize the muscle regulatory factor MRF4 and determine how it functions in establishing and maintaining stable muscle cell lineages. MRF4 is a member of the basic/helix-loop-helix (b/HLH) protein family that also includes the muscle regulatory factors MyoD, myogenin and Myf-5. Expression of an MRF4 cDNA in fibroblasts converts the cells to stable muscle cell lineages that can be maintained as undifferentiated myoblasts or that can be induced to transcriptionally active expression of the alpha-actin, myosin, tropomyosin, troponins C, I and T, and M-creatine kinase genes. MRF4 expression initially is detected in the early somites of developing embryos, later in differentiated myofibers and is the predominant muscle regulatory factor in the adult organism, suggesting that MRF4 has an essential role in both establishing and maintaining the phenotype of the mature muscle fiber. MRF4, when complexed with the ubiquitously expressed b/HLH protein, E12, recognizes and binds to an E-box DNA consensus sequence which is found within the enhancer elements of most muscle-specific genes. These specific protein:DNA interactions are required for MRF4 to trans-activate expression of the contractile protein genes. Although MRF4 clearly acts as a tissue-specific transcription factor, it is unknown how the MRF4 protein is regulated developmentally, how it functions at the molecular level or how MRF4 interacts with additional transcription factors to produce a functional muscle fiber. to decipher how the MRF4 protein regulates skeletal myogenesis requires a detailed structure/function analysis. Towards this end, the DNA binding, hetero-dimerization and trans-activation properties of MRF4 in responsive and non-responsive cell lines will be examined, as well as how peptide growth factors and protein phosphorylation events regulate MRF4 activity. In addition, proteins that interact with MRF4 to regulate its muscle-specific activity will be identified and isolated. This comprehensive approach toward characterizing the MRF4 protein will generate novel information on how the muscle regulatory factor gene family operates to control skeletal muscle development. These studies also are designed to provide a better understanding of how transcription factor activities are modulated in this unique developmental system.