| Summary: | 碩士 === 國立中央大學 === 生命科學系 === 102 === Myogenic regulatory factors (MRFs), which include MyoD, Myf5, MRF4 and Myogenin, are the master regulators of skeletal myogenesis. During embryogenesis, notochord and neural tube secrete Shh and Wnts to induce MyoD and Myf5 expression in somites to initiate myogenesis. Wnts are extracellular glycolipoproteins regulating several key developmental processes, such as proliferation, differentiation, asymmetric division, patterning, and cell fate determination, and they have three downstream pathways: canonical, non-canonical, and Wnt/ Ca2+ pathway. Recent researches had shown that upstream responsive elements of MyoD regulate its promoter activity, but how Wnts affect this regulation has not been established. Our previous data had shown that Wnt3a could regulate MyoD expression and its promoter activity when distal enhancer (-20~ -25k) was attached. To further identify how Wnt3a regulates MyoD expression, firstly MyoD reporter constructs containing various upstream regions were established for narrowing down the possible Wnt3a target site. Our data showed that an upstream region (-8~ -9k), whether linked to distal enhancer (~5k) or not, could mediate Wnt3a stimulated MyoD promoter activity. Secondly, using specific inhibitors of each pathway downstream to Wnt3a, we found that multiple pathways, including canonical pathway mediated by β-catenin, were mediating Wnt3a effect on MyoD expression. These observations were further confirmed by over-expressing dominant negative effectors of Wnt signaling pathways. Finally, the physiology relevance of β-catenin regulated MyoD expression in vivo was assessed by Chromatin Immunoprecipitation (ChIP) assay, and we found strong binding of β-catenin to multiple sites in the MyoD upstream regulatory region. Taken together, these results have soundly demonstrated the direct regulation of the MyoD expression by Wnt3a signaling through targeting multiple upstream cis-elements.
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