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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/65047

    Title: 探討 Wnt3a調控 MyoD表現機制;Defining the pathways and responsive elements mediating Wnt3a activated MyoD promoter activity
    Authors: 潘語之;Pan,Yu-chih
    Contributors: 生命科學系
    Keywords: 啟動子;肌肉;肌肉形成;MyoD;promoter;Wnt3a;muscle;β-catenin;myogenesis
    Date: 2014-07-29
    Issue Date: 2014-10-15 14:39:00 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 肌肉轉錄調控因子 (MRFs),其中包含 MyoD、Myf5、MRF4、Myogenin,是調控骨骼肌發育的主控因子。胚胎發育過程中,脊索與神經管分泌 Shh及 Wnts誘導體節的 MyoD與 Myf5表現,使肌肉開始發育。Wnts為分泌至胞外的一群醣蛋白,調控著增生、分化、不對稱分裂、決定細胞命運…等發育過程。Wnt訊息傳導有三條路徑:典型、非典型與Wnt/鈣離子路徑。近來的研究顯示,MyoD上游反應區域會調控其啟動子活性,但對於 Wnts調控 MyoD的轉錄活性機制尚未建立。本實驗室先前的數據指出,Wnt3a調控 MyoD的表現及其啟動子活性,需倚靠啟動子上游-20~-25 kb的遠端增強子 (Distal Enhancer)。為了進一步確認 Wnt3a如何調控 MyoD表現,首先,我們構築 MyoD 報導質體與啟動子上游各個不同區域的質體,欲建立一個系統,利於我們縮小區域並尋找出 Wnt3a的目標位。結果顯示,上游區域 (-9~-8 kb),不論是否與遠端增強子連結在一起,皆可以經由 Wnt3a的刺激而活化 MyoD啟動子的活性。其次,我們利用具特異性的抑制劑,欲抑制 Wnt3a下游傳導路徑,找出可能的傳導路徑。結果發現幾個路經皆參與其中,包括典型的Wnt/β-catenin路徑。Wnt3a藉由 β-catenin的刺激影響 MyoD的表現。這些觀察到的結果,我們藉由大量表現 Wnt訊息傳導下游路徑的顯性抑制影響因子 (Dominant negative effector),進一步得到證實。最後,我們利用了染色質免疫沉澱法,為探究在體內 β-catenin調控 MyoD表現的生理意義。結果發現,在 MyoD上游調控區域具有多個 β-catenin結合位,且結合的能力很強。總而言之,這些結果徹底地證明 Wnt3a訊號經由結合多個上游區域直接調控 MyoD表現。;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.
    Appears in Collections:[生命科學研究所 ] 博碩士論文

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