探討大量表現 FoxO6 對肌肉終極分化的影響以及尋找 FoxO6 蛋白質在 PGC-1 alpha 啟動子上的結合位 ; Effects of FoxO6 over-expression on terminal myogenic differentiation and defining the FoxO6- binding site in the PGC-1 alpha promoter

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Title:	探討大量表現 FoxO6 對肌肉終極分化的影響以及尋找 FoxO6 蛋白質在 PGC-1 alpha 啟動子上的結合位;Effects of FoxO6 over-expression on terminal myogenic differentiation and defining the FoxO6- binding site in the PGC-1 alpha promoter
Authors:	李冠緯;Kuan-Wei Lee
Contributors:	生命科學研究所
Keywords:	肌肉終極分化;PGC-1 alpha;FoxO6
Date:	2010-07-29
Issue Date:	2010-12-08 14:41:50 (UTC+8)
Publisher:	國立中央大學
Abstract:	Forkhead box O (FoxO)轉錄因子能夠調節細胞中各種生理功能，包含細胞週期、葡萄糖代謝、壓力反應、細胞分化以及細胞的死亡與存活機制等。在2003年發現了一個新的Forkhead家族成員-FoxO6。由於它缺少了一些重要的胺基酸使得進出細胞核的能力受到影響，其中最重要的是缺少了C端保守的PKB可磷酸化胺基酸，這個位置是FoxO成員主要移出核的調控區域，也因此FoxO6會比其它FoxO成員更容易滯留在細胞核中。雖然目前研究發現，FoxO6主要表現在腦部組織，但在肌肉中也有發現FoxO6的表現，而且FoxO6對於骨骼肌的功能目前尚未清楚，因此FoxO6對骨骼肌的影響成為我們主要的研究目標。肌肉中另一個重要因子-PGC-1 alpha，它是一個PPAR-gamma?的輔助轉錄因子能夠與 PPAR-gamma 相互作用，在粒線體的代謝中更扮演一個非常重要的角色，若是在肌肉中大量表現PGC-1 alpha?會促使肌肉的型態從TypeII肌纖維變成TypeI肌纖維。從我們的研究中發現大量表現FoxO6的穩定細胞株C2C12-mFoxO6比起控制組更能降低肌管的數量及大小，這表示FoxO6能夠抑制肌肉的分化。於是我們推測也許會干擾細胞週期來影響肌肉細胞的分化。然而在流式細胞儀分析實驗中，confluent時期的C2C12-mFoxO6也能與控制組一樣停留在G0/G1期。此外，我們發現了過量表現FoxO6會抑制PGC-1 alpha?基因的表現。於是我們推測FoxO6可能可以直接調控PGC-1 alpha?啟動子的轉錄活性。在啟動子活性試驗中，發現PGC-1 alpha 啟動子的活性會受到FoxO6的調控，而利用EMSA以及Footprinting也證實了FoxO6能夠直接結合在PGC-1?alpha 啟動子上，而在未來，我們希望能利用chip試驗來加以印證FoxO6與PGC-1 alpha?啟動子的關係。 Forkhead box O (FoxO) transcription factors could regulate many cellular functions, including cell cycle, glucose metabolism, stress response, cell differentiation, cell death and survival. FoxO6, one of the forkhead family, was discovered in 2003. It was different from other FoxO factor because FoxO6 remained mostly nuclear due to its impaired shuttling ability . It lacked the conserved C-terminal PKB motif, which was the cause of the shuttling impairment. Although FoxO6 is majorly expressed in developing brain, it is also expressed in muscle and its function in skeletal muscle was unknown. PGC-1 alpha??was a transtcription coactivator that interacted with PPAR-gamma?and?played a key role in mitochondrial metabolism. When PGC-1 alpha?was?overexpressed in muscle, it transformed type2 myofiber into type1 myofiber. Our study had discovered that C2C12-mFoxO6 cell line could decrease the number and size of myotube compared with C2C12-control. We supposed that FoxO6 may be interfere with cell cycle to inhibit myogenic differentiation. However in flow cytometer assay, confluent C2C12-mFoxO6 myoblast also could majorly stay at G0/G1 phase during cell cycle, it meaned that C2C12-mFoxO6 myoblast also had normal cell cycle exit. We also found that overexpression of FoxO6 inhibited expression of PGC-1 alpha gene in confluent C2C12 myoblast. Thus, we supposed that FoxO6 may regulate PGC-1 alpha promoter activity. By using reporter assay we demonstrated that FoxO6 could inhibit the PGC-1?alpha?promoter activity. Then, we supposed FoxO6 may bind directly to PGC-1?alpha promoter. By EMSA and footprinting we demonstrated that FoxO6 really could bind to PGC-1?alpha?promoter. In the future, we hope to confirm the binding site by chromatin immunoprecipitation assy.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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