The proposed research examines the molecular mechanisms by which coordinate gene activation is regulated during the embryonic formation, maturation, and diversification of mammalian skeletal muscle tissue. In recent years, tremendous progress has been made towards understanding the molecular mechanisms that activate muscle-specific genes as committed myoblasts undergo the transition to terminally differentiated myotubes. A fundamental, but much less well defined, aspect of myogenesis is the maturation of muscle cells into specific types of myofibers. During the past NSF support period, the PI characterized the gene encoding the human slow-twitch isoform of troponin I (TnIs) and has shown that multiple cis-acting components are involved in the cell type-expression of the TnIs promoter; an upstream muscle- specific enhancer, a second muscle-specific enhancer within intron 1, and a muscle-specific minimal promoter. In conjunction with collaborators, the PI demonstrated that a TnIs promoter/CAT construct exhibits appropriate muscle fiber type-specific expression in transgenic mice, but preliminary results have indicated that the transgene (which lacks the intron enhancer) is aberrantly regulated during embryogenesis. The hypothesis to be tested is that gene regulation during early and late stages of muscle development requires separate regulatory elements which bind different combinations of trans-acting factors; the upstream elements may play a major role in the innervation dependent restriction of TnIs gene expression to slow twitch muscle fibers and the downstream sequences, such as the intron 1 enhancer, may play a major role in earlier stages of myofiber formation. In order to map the upstream sequences conferring fiber type-specific gene expression, the PI established an in vivo muscle injection assay with which to study differential gene expression in fast versus slow twitch muscles of adult rats. This system enables the PI the unique opportunity to un derstand the molecular mechanisms by which fiber type specificity is regulated. The PI will use the in vivo injection assay to delineate cis- acting components involved in the fiber type-specific expression of the TnIs gene. Once such cis-elements are narrowly defined in vitro mutagenesis studies will be used to monitor the consequences of modifying these fiber- specific regulatory elements. Additional lines of transgenic mice will be produced in order to study the role of these sequence elements in the regulation of gene expression during muscle development and maturation. The expression of transgene constructs in embryonic and adult mice will be monitored by reporter gene activity assays along with Northern blot, immunohistochemical, and in situ hybridization analyses. The following questions will then be addressed: 1) What are the cis-acting sequences and trans-acting factors that restrict TnIs gene expression to slow-twitch fibers in adult skeletal muscle, and how might these be related to the components governing muscle-specific expression in vitro? 2) How do "muscle-specific" and "fiber-specific" regulatory elements contribute to the spatial and temporal control of gene expression during muscle development and maturation in vivo? 3) Do the upstream and intron enhancers mediate separate aspects of TnIs gene regulation in primary versus mature myofibers?
