The myoD regulatory gene family encode skeletal muscle-specific transcription factors that serve critical functions in the establishment and differentiation of skeletal muscle lineages. The long-term objective of the proposed research is to understand the cis and trans transcriptional control mechanisms that govern the expression of the human myoD gene during embryonic development. The recent cloning of the myoD enhancer, which directs the embryonic expression of myoD (Goldhamer et al., Science 256:538), now makes this analysis possible. The myoD enhancer will be systematically mutagenized and expression analyzed in transgenic mice. These studies will: (1) identify cis-acting DNA sequences required for muscle-specificity; (2) determine whether this specificity is governed by positive or negative transcriptional control; (3) test the hypothesis that myoD is regulated by distinct mechanisms in different skeletal muscle lineages of the embryo; (4) determine whether transcriptional activation and maintenance of expression are mediated by distinct mechanisms. Mutagenesis of the conserved E-box motifs within the enhancer will determine if direct cross- and auto-regulatory interactions between members of the myoD family regulate myoD expression in vivo. Analysis of enhancer transgene expression in myoD deficient mice will specifically test the hypothesis that MyoD protein regulates the myoD gene in a positive auto-regulatory feedback loop. The function of DNA methylation in repressing myoD in non-muscle tissues, suggested by previous cell culture experiments, will be tested by comparing the methylation status of the enhancer in various mouse tissues using PCR-based genomic sequencing, and by functional analysis of the conserved CpG dinucleotides. Finally, expression cloning of lambda-gt11 libraries with nuclear factor binding sites and mutation-sensitive cis elements within the enhancer will be used to clone trans-acting factors that regulate myoD. Expression and functional analyses of these """"""""upstream"""""""" factors will begin to define early molecular events associated with the establishment of skeletal muscle lineages. In addition to regulating skeletal myogenesis in consort with the other myogenic factors, inappropriate expression of myoD during embryonic development causes developmental defects and death. Clearly, normal growth and development requires the precise regulation of myoD. The present studies will contribute substantially to an understanding of how this precision is effected.
