| Research Abstract |
1) In order to analyze the roles of individual members of myogenic regulatory factors in myogenesis, we have investigated their effects on the transcription driven by the promoter of the chicken myosin alkali light chain (MLC1/3) gene. CMD1(MyoD) or c-myogenin, but cMRF4 activated transcription of reporter plasmid containing 3.4kb upstream region of MLC1 gene in primary fibriblasts. The activation by MyoD or myogenin was dependent on the existence of a muscle-specific enhancer located from -2096 to -1743. The distal half of the enhancer, containing a pair of E-boxes, E1 and E2, was found to be a MyoD specific enhancer. In contrast, the activation by myogenin was dependent on the existence of the proximal half of the enhancer, containing three E-boxes, E3,E4,E5. In their E-boxes, the proximal E-box, E5 was essential for trans-activation by myogenin. Although MyoD and myogenin bound to E5 with similar affinity to each other, E5 and its falnking sequence worked as a myogenin-specific enhancer. From the analysis of the effect of chimeric proteins of MyoD and myogenin, the region(s) outside of the basic-helix-loop-helix (b-HLH) domain of myogenin was involved in the specificity of the myogenin-specific enhancer.
2) To understand regulatory mechanisms which govern transcriptional activation of the myogenin gene, transgenic mice bearing the lacZ gene driven by the upstream region of the myogenin gene were generated. Stereoscopic visualization of LacZ positive cells of these transgenic mouse embryos revealed that the upstream region of the myogenin gene conferred its transcriptional activation in cells of the skeletal muscle lineages in somites, limb buds, and viceral arches.
3) To investigate the molecular function of myogenin gene during muscle development, myogenin gene was desrurpted by homologous recombination. The analysis of effects of myogenin gene knock out is now in progress.
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