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研究生: 張心瑜
Hsin-yu Chang
論文名稱: PGC-1α 與 Stra13 間之交互作用
The Interaction between PGC-1α and Stra13
指導教授: 陳盛良
Shen-Liang Chen
口試委員:
學位類別: 碩士
Master
系所名稱: 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度: 98
語文別: 中文
論文頁數: 76
中文關鍵詞: PGC-1αStra13
外文關鍵詞: PGC-1α, Stra13
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  • PPAR-γ coactivator-1 (PGC-1α) 是DNA轉錄因子的共同調控因子,參與在能量代謝的調控機制中,並且與肌肉纖維型態轉換有關。 當細胞受到外在環境的刺激,例如:冷,PGC-1α會被大量誘導表現。在之前已被證實PGC-1α可以和PPARγ及Mef2c共同調控其下游及自身基因的活化。 Stra13在轉錄層次上大部分扮演抑制者的功能,它是一個basic helix loop helix (bHLH)的轉錄因子。這一類的轉錄因子利用鹼性的區段與DNA的固有序列結合,Stra13屬於bHLH轉錄因子的E group,但是結合的DNA固有序列卻是B group 結合的位置。 在本實驗室的研究中發現,Stra13可以抑制MyoD對於PGC-1α的transactivation的能力。在我們的研究當中,想要去了解PGC-1α和Stra13兩個蛋白質之間的交互作用,以及兩者交互作用對於PGC-1α和Stra13下游調控的基因的影響。在cell free的實驗方面,已知道PGC-1α和Stra13會有很強的結合能力,我們利用GST pull down assay的方式去觀察到PGC-1α主要利用1-128 a.a.的位置和Stra13交互作用,而Stra13則是利用1-135 a.a.的位置與PGC-1α交互作用。 接著在in cell的實驗方面,將針對PGC-1α下游的調控基因,藉由nuclear respiratory factor和nuclear recptor活化後,觀察PGC-1α和Stra13交互作用的影響。利用同樣的方式觀察Stra13下游基因會不會受到影響。在未來希望能建立PGC-1α-flag穩定表現之細胞株,然後以co-immunoprecipitation的方式,觀察PGC-1α與Stra13在in cell的交互作用情形。


    PPAR-γ coactivator-1 (PGC-1α) serve as a coregulators of DNA-binding transcription factors controlling energy metabolism. It is implicated in the muscle fiber type- switching, response to cold exposure and other cellular energy metabolisms. Interestingly, it has also been shown that PGC-1α can coactivate PPARγ- and Mef2c-dependent activation of its own gene. Stra13 is a basic helix-loop-helix (bHLH) containing transcriptional repressor of the E group but binds to the consensus motif as B group factors. In our recent study, we found that Stra13 can repress MyoD transactivation of PGC-1α promoter activity. In this study, we are interested in the interaction between PGC-1α and Stra13 on PGC-1α- and Stra13-dependent genes. In cell-free system, pull down assay indicated that PGC-1α binds to Stra13 strongly. We also defined the regions of PGC-1α (1-128) amino acid play an important role in the interaction with Stra13. And Stra13 (1-135) amino acid is the major region which interacts with PGC-1α. To go a step further, our study is to find out whether the interaction between PGC-1α and Stra13 affects the activation of PGC-1α-dependent genes by nuclear respiratory factor and nuclear receptors. Similarly, it is also of interest to know the response of Stra13 target genes to the presence of PGC-1α. In the future, we will use co-immunoprecipitation assay as an in-cell system to reveal that their interaction.

    中文摘要 I ABSTRACT II 誌謝 III 縮寫與全名對照表 VIII 一、緒論 1 Ⅰ 肌肉的起源與生成 1 Ⅱ The PGC-1α family 2 a PGC-1α 2 b PGC-1β 4 c PRC 4 Ⅲ Stra13 5 Ⅳ 研究動機與目的 6 二、材料與方法 8 Ⅰ 實驗材料 8 1 細胞株 8 1-1 細胞培養 8 2 菌株 8 2-1 菌株培養 8 2-2 菌株的保存 8 Ⅱ 質體構築 8 1 pM-PGC-1α表現質體 (expression plasmid) 建構 8 1-1 插入DNA (PGC-1α)的製備 9 1-11 聚合鏈鎖反應Polymerase Chain Reaction (PCR) 9 1-12 聚合鏈鎖反應產物的修飾 9 1-13 插入 (Insert) DNA的純化 9 1-2 載體DNA的製備 10 1-21 載體DNA限制的剪切 11 1-22 載體 DNA 的 5’端去磷酸根反應 11 1-23 載體DNA的純化 11 1-24 接合反應 ( Ligation ) 11 1-25 篩選 (Screening) 11 2 pcDNA3-PGC-1α (1-186)質體的建構 12 2-1 純化步驟 12 3 pcDNA3-PGC-1α (1-292) 質體的建構 13 4 pcDNA3-PGC-1α (1-403) 質體的建構 13 5 pcDNA3-PGC-1α (1-66) 質體的建構 13 6 pcDNA3-PGC-1α (64-128) 質體的建構 13 7 pcDNA3-PGC-1α (128-196) 質體的建構 13 8 pcDNA3-PGC-1α (1-128) 質體的建構 13 9 pcDNA3-PGC-1α (64-196) 質體的建構 14 10 pcDNA3-PGC-1α (196-799) 質體的建構 14 11 pcDNA3-Stra13 (1-135)質體的建構 14 12 pcDNA3-Stra13 (1-194)質體的建構 14 13 pcDNA3-Stra13 (1-288)質體的建構 14 14 pcDNA3-Stra13 (1-51)質體的建構 14 15 pcDNA3-Stra13 (1-108)質體的建構 14 14 pcDNA3-Stra13 (52-108)質體的建構 14 16 pcDNA3-Stra13 (108-135)質體的建構 14 Ⅲ RT-PCR 15 1 Total RNA 的抽取 15 2 反轉錄反應 (Reverse Transcription) 15 3 聚合鏈鎖反應 ( Polymerase Chain Reaction, PCR ) 16 Ⅳ Real Time PCR 定量實驗 16 Ⅴ 轉染實驗 17 1 哺乳細胞雙雜交轉染實驗 17 1-1細胞培養 17 1-2轉染作用 (Transfaction) 17 1-3螢火蟲冷光活性方法 (Luciferase Activity Assay) 17 2 過渡性轉染實驗 17 Ⅵ 蛋白質標定 18 Ⅶ 蛋白質純化 18 Ⅷ GST pull down assay 19 三、實驗結果 21 1 利用mammalian two-hybrid assay觀察PGC-1α與Stra13兩個蛋白質在in cell system 中交互作用的情形 21 2 PGC-1α 主要以 (1-186 aa) 與MBP-Stra13 binding 22 3 PGC-1α (1-128) 與MBP-Stra13結合的能力最強 23 4 Stra13不同長度之deletion對GST-PGC1α結合能力 24 5 比較C3H10T1/2與C2C12兩株細胞中PGC1α表現量的差異 25 7 Overexpression PGC1α及Stra13時,對Vascular Cell Adhesion Molecule -1 (VCAM-1) promoter的影響 27 8 pPYCAGIP-PGC-1α-flag質體的建構 27 四、實驗討論 29 五、圖表 33 圖一、 pM-Stra13與pcDNA3-PGC-1α對TK-MH-100x4-LUC活性的影響 34 圖二、 PGC-1α CDS利用酵素分成不同片段示意圖 35 圖三、 觀察PGC-1α不同C端deletion clones與Stra13在in vitro的結合情形 36 圖四、 PGC-1α (1-186)之間deletion clones與PGC-1α C端196-799 示意圖 37 圖五、 觀察PGC-1α(1-186)之間的deletion clones與Stra13在in vitro的結合力 38 圖六、 PGC-1α (1-186)之間deletion clones與PGC-1α C端 (196-799) 示意圖 39 圖七、 觀察PGC-1α (1-186)之間的deletion clones與Stra13在in vitro的結合力 40 圖八、 純化GST-PGC-1α-beads 41 圖九、 Stra13 C端deletion clones示意圖 42 圖十、 Stra13 C端不同deletion clones與GST、GST-PGC-1α結合情形 43 圖十一、 Stra13 (1-135)更小片段的deletion clones示意圖 44 圖十二、 Stra13 (1-135)之間不同deletion clone對GST、GST-PGC-1α結合情形 45 圖十三、 純化 MBP-Stra13 (108-135)、MBP-Stra13 (52-135) 46 圖十四、 PGC-1α及Stra13對IAP promoter活性影響及比較C2C12、 48 C3H10T1/2兩細胞株中,PGC-1α表現量 48 圖十五、 PGC-1α及Stra13對promoter活性之影響 50 圖十六、 過量表達PGC-1α及Stra13時對VCAM-1 promoter轉錄活性影響 51 圖十七、 py-PGC1α-flag質體建構 52 六、參考文獻 53 七、附錄一 55 <圖一> 55 <圖二> 56 <圖三> 57 <表一> 58 <表二> 58 <表三> 59 八、附錄二 60 <PCR primer對照表> 60 九、附錄三 61 I 溶液及試劑配方 61 II 藥品試劑 63 III 酵素和限制 63

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