IDENTIFYING REGULATORY ELEMENTS IN THE TPNC41C ENHANCER REGION RESPONSIBLE FOR FIBER-SPECIFIC EXPRESSION IN DROSOPHILA MELANOGASTER
Sara Maes, Maria Chechenova, Richard Cripps.
University of New Mexico, Albuquerque, NM.
Similar to skeletal muscles in vertebrates, the somatic muscle of Drosophila is composed of different fiber types that vary in their structural and functional properties. An example of such muscles are the indirect flight muscles (IFMs) and jump muscles of the fly thorax. Although the early development of muscle tissue has been thoroughly studied, the mechanisms by which muscle fibers initially become specified into their different types remains unclear. To further our understanding of this mechanism, we studied the transcriptional regulation of the jump muscle-specific troponin gene, TpnC41C, of D. melanogaster. Analysis of the highly conserved genomic region directly upstream of the gene revealed a 300 bp enhancer sufficient for its proper expression. Two sequences within this region were analyzed as possible regulatory sites essential for activation of TpnC41C’s tissue-specific expression. Using site-directed mutagenesis, we developed reporter plasmids with individual and combined mutations of these sequences within the TpnC41C enhancer placed upstream of a LacZ gene. Flies containing genomic insertion of these plasmids were examined for expression of the reporter protein β-galactosidase. Reporter activity tested through immunohistochemistry on cryosections and in vitro enzymatic assays showed that mutations in these sites greatly reduced enhancer activity. Of the two sites, one was confirmed through in vitro binding assays to specifically bind Myocyte Enhancer Factor-2. Identification of the transcription factors necessary for specific gene expression in model organisms such as Drosophila melanogaster provides us with knowledge of the evolutionarily conserved regulatory pathways of muscle formation in all animals, including mammals.