This is a renewal application to study the transcriptional regulation of both the type I and IIB MHC genes in skeletal muscle. In previous work the investigators have shown that both of these genes demonstrate a unique pattern of altered expression in skeletal muscle in response to a variety of physiological stimuli. Both genes are highly regulated and, for the most part, in opposite directions by transcriptional/translational processes in response to alterations both in loading conditions and in thyroid state. Both require the presence of intact innervation for optimal expression. The primary goal of the present application is to functionally characterize the promoter regions for both genes and specifically to identify cis-regulatory and corresponding trans-acting factors which are responsible for the regulation of these two genes in different types of skeletal muscle. The experiments will involve the following strategies: the first is to study in vivo transcriptional activities of both type I and IIB MHC promoter by using reporter gene assays following direct skeletal muscle injection of chimeric plasmid DNA in which different portions of the promoter are linked to a reporter gene. This approach will include deletional analysis of each promoter and will also include the testing of constructs containing mutations at specific regulatory site, rearrangements of proposed enhancer sequences and cis-acting elements thought to be regulating the promoter activity. Dnase footprinting experiments to identify regulatory sequences, gel mobility shift assays to detect DNA interactions will also be performed as will strategies to either over express or reduce expression of putative transacting factors in vivo. These approaches will delineate the molecular mechanism responsible for the regulation of these genes in the in vivo setting under a wide range of physiologic conditions.
Showing the most recent 10 out of 62 publications
