探討 Wnt3a調控 MyoD表現機制

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姓名

潘語之(Yu-chih Pan) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討 Wnt3a調控 MyoD表現機制
(Defining the pathways and responsive elements mediating Wnt3a activated MyoD promoter activity)

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 啟動子
★ 肌肉
★ 肌肉形成

關鍵字(英)

★ MyoD
★ promoter
★ Wnt3a
★ muscle
★ β-catenin
★ myogenesis

論文目次

目錄
中文摘要 VIII
Abstract IX
誌謝 X
一、緒論 1
I. 肌肉發育(Muscle development) 1
胚胎發育 (Embryogenesis) 1
體節形成 (Somitogenesis) 2
肌肉形成 (Myogenesis) 2
II. 肌肉轉錄調控因子 (Myogenic regulatory factor, MRF) 3
生肌決定因子 (Myogenic Determination Factor1, MyoD) 3
MRF與肌肉發育之關係及其調控機制 4
III. Wnt signaling pathway 5
起源 5
訊息傳導路徑 6
Wnt與肌肉發育之關係 7
IV. 研究動機與目的 8
二、材料與方法 9
2-1細胞株 (Cell Line) 9
2-1-1選用細胞株 9
2-1-2穩定細胞株製備 9
2-1-3細胞培養 9
2-2質體構築 (Cloning) 10
2-2-1選用菌株 10
2-2-2菌株培養 10
2-2-3質體構築 10
2-2-4質體構築流程圖 16
2-3過渡性轉染作用 (Transient transfection) 21
2-4螢火蟲冷光活性測方法 (Luciferase Activity Assay) 22
2-5逆轉錄聚合酶鏈式反應 (Reverse Tanscription Polymerase Chain Reaction, RT-PCR) 22
2-5-1 Total RNA製備 22
2-5-2反轉錄作用 (Reverse transcription) 23
2-5-3聚合酶鏈反應 (Polymerase Chain Reaction , PCR ) 23
2-5-4即時聚合酶鏈反應 (Real Time Polymerase Chain Reaction , RT-PCR ) 24
2-6西方墨點法 (Western Blot) 24
2-6-1 Total protein lysate製備 25
2-6-2聚丙烯醯胺凝膠電泳 (SDS-polyacrylamide Gel Electropheresis) 25
2-6-3轉印 (Transfer) 25
2-6-4 Blocking 及抗體 (Antibody) 辨識 26
2-6-5 Antibody蛋白質脫附 (Stripping) 26
2-7染色質免疫沉澱 (Chromatin immunoprecipitation assay, CHIP) 27
三、實驗結果 30
3-1 驗證Wnt3a確實分泌至所收取的medium及其功能性 30
3-2 在不同時期的肌肉細胞，皆無 Wnt3a蛋白質表現 31
3-3 PME24521 (#9)與 PME2421 (#10)的片段與 PME組別相比沒有顯著的差異 32
3-4 Wnt canonical and non- canonical pathways皆會影響MyoD promoter之轉錄活性 33
3-5在in vivo的情況下，β-catenin對於 MyoD promoter上游調控區域具有很強的結合能力 34
3-6 大量表現 β-catenin有效的使 C2C12細胞在 CMB及 MT時期 MyoD mRNA表現量提高 35
3-7 MyoD promoter與enhancer間的距離會影響其轉錄活性 36
3-8 L fragment非經由 Wnt canonical and non- canonical pathways調控 MyoD gene轉錄活性 36
四、討論 38
4-1 MyoD Ehancer與 Promoter上游-9k~-8k區域，經由何種方式活化 MyoD promoter轉錄活性 38
4-2 Enhancer對於 MyoD promoter調控 39
4-3 Wnt3a調控 MyoD上游區域之機制在生理上有何意義 40
4-4 Serum starvation對於 Wnt3a secretion之影響 41
五、圖表 42
圖一、 293T-Py-Wnt3a的培養液含有 Wnt3a蛋白質且具有功能性 44
圖二、構築不同片段小大之 Enhancer至 Pstable-MyoD6.0-Luc質體 45
圖三、處理 293T-Py-Wnt3a後，PME#9、PME#10與 PME相比之轉錄活性並無顯著差異 47
圖四、 Wnt canonical and non- canonical pathways皆會影響 MyoD promoter之轉錄活性 50
圖五、在in vivo的情況下，β-catenin 對於MyoD promoter上游調控區域具有很強的結合能力 52
圖六、大量表現 β-catenin有效的使 C2C12細胞在 CMB及 MT時期 MyoD mRNA表現量提高 55
圖七、MyoD promoter與enhancer間的距離會影響其轉錄活性 56
圖八、 PM-L非經由 Wnt canonical or non- canonical pathways調控 MyoD轉錄活性 58
六、參考文獻 61
七、附錄 65
附錄一、 65
附錄二、 68
附錄三、 70
附錄四、 72
附錄五、 77
附錄六、 78
附錄七、 81
附錄八、 82
附錄九、 83

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指導教授

陳盛良(Shen-liang Chen)

審核日期

2014-7-29

推文