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研究生:陳明容
研究生(外文):Ming-Rong Chen
論文名稱:細胞分裂期中EZH2磷酸化作用的研究
論文名稱(外文):The functional role of mitotic phosphorylation of EZH2(Enhancer of Zeste 2)
指導教授:查岱龍查岱龍引用關係
指導教授(外文):Tai-Lung Cha
口試委員:黃世明蕭培文
口試委員(外文):Shih-Ming HuangPei-Wen Hsiao
口試日期:2011/06/29
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:77
中文關鍵詞:細胞分裂磷酸化染色體周邊層
外文關鍵詞:EZH2mitosisphosphorylation
相關次數:
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EZH2 為polycomb repressvie complex2(PRC2)的核心酵素,在組蛋白H3 進行三重甲基化作用抑制基因表現,生物功能則與附基因體印記的維持、細胞命運決定與癌化惡性生長有關。。對於多細胞生物而言,在時間與空間層次細緻的調節EZH2 活對其生長發育相當重要,了解EZH2 的調控機制是幹細胞生物學與癌症生物學的關鍵。後轉譯修飾作用對於蛋白質功能的調節非常重要,因此我們將由EZH2 的後轉譯修飾作用切入調控機制的研究。
EZH2 受到與細胞週期調控相關的RB/E2F 訊息傳遞路徑的調控,又控制著一些調控細胞周期的基因,例如:INK4a/ARF 以及CDKN1C;且多處EZH2 磷酸化作用發生在細胞分裂期,表示EZH2 可能參與在細胞分裂的機制。近來發現,CDK1 可在EZH2 的第345 與第487 酥胺酸進行磷酸化作用,但其生物亦亦仍待更多研究。此外,EZH2 與其受質H3K27三重甲基化標誌出現於分裂期染色體中,協助適當的附基因體記憶維持;然而,目前的證據還不足以說明EZH2 在細胞分裂期中的功能。在本研究中,發現在細胞分裂初期,磷酸化EZH2 聚集於染色體周邊的獨特的分布位置,顯示EZH2 在細胞分裂過程中具有不同於甲基轉移酵素的嶄新功能。
EZH2, the catalytic core consisted in Polyocmb repressive complex 2(PRC2), plays an important role in epigenetic memory maintenance, cell fate determination, and malignant progression. PRC2 marks chromatin
with histone H3 lysine tri‐methyation, followed subsequent transcriptional repression. EZH2 and PRC2 can target genes as OCT4, Nanog, Pax7, HOX cluster for development; VASH1 for angiogenesis; SLIT2 and DAB2IP
for cancer metastasis; and INK4a/ARF for cell cycle control. Delicately spatial and temporal control of EZH2 activity is essential to orchestrating development of multicelluar organism, thus, knowing the biological events regulating EZH2 is pivotal to stem cell biology and cancer biology.
To further investigate the regulatory mechanism of EZH2, dynamic post‐translational modification of EZH2 was took into considerations. First of all, EZH2 regulates a group of genes associated with cell cycle, such as INK4a/ARF and CDKN1C. And EZH2 is regulated by RB/E2F pathway, which controls cell cycle. Last and the most significantly, EZH2 is phosphorylated at multiple sites during mitosis, implying an active involvement. Recently, Cyclin‐dependent kinase 1(CDK1) was identified as a potent EZH2 kinase, which phosphorylate EZH2 at T487 and T350 in mitotic stage vastly, but the biological meanings need further researches. In addition, EZH2 and H3K27me3 marker associated with mitotic chromosome
contributed to proper epigenetic memory; however, evidences to date are not sufficient to propose a scenario EZH2 plays in mitosis. In the study, we identified a unique structural localization of phorphoylated EZH2 during mitosis, which suggests a novel function distinct from methyltransferase
in cell cycle progression.
正文目錄 .............................................................................. I
表目錄 ............................................................................... III
圖目錄 ............................................................................... IV
中文摘要 ............................................................................. V
英文摘要 ............................................................................ VI
第一章 緒言 ....................................................................... 1
第一節、Polycomb Repressive Complex 2(PRC2)之簡介 ........................... 1
第二節、EZH2 與癌症發展 ........................................................................ 2
第三節、EZH2 與發育生物學 .................................................................... 4
第四節、EZH2 的後轉譯修飾作用 ............................................................ 5
第五節、PRC2、EZH2 與細胞分裂 ............................................................ 7
第六節、染色體周邊層(perichromosomal layer) ...................................... 9
第七節、研究背景與目的 ........................................................................ 10
第二章 材料與方法 .......................................................... 12
第一節、細胞株 ....................................................................................... 12
第二節、細胞培養與轉染 ........................................................................ 12
第三節、細胞蛋白質樣本製備 ................................................................ 13
第四節、免疫沉澱法(Immunoprecipitation) ........................................... 14
第五節、膠體電泳 ................................................................................... 15
第六節、膠內水解(In‐gel digestion)與質譜儀胜肽樣本製備 ................. 16
II
第七節、西方墨點法(Western blotting) .................................................. 18
第八節、表現質體(plasmids) ................................................................... 19
第九節、免疫螢光染色(Immunofluorescence Staining) ......................... 19
第十節、染色質分離(chromatin fractionation) ....................................... 21
第十一節、DNAseI 水解酵素測試(DNAseI sensitivity assay) .................. 22
第三章 結果 ..................................................................... 24
第一節、以磷酸化蛋白體學方式確認EZH2 的磷酸化位點 .................. 24
第二節、磷酸化EZH2(EZH2‐PT487)出現於分裂期染色體周邊 ............. 25
第三節、EZH2‐pT487 出現於分裂期前期、中期與後期 ....................... 27
第四節、EZH2‐PT487 屬於染色體周邊層蛋白 ....................................... 28
第五節、EZH2‐PT487 與染色質的相對關係 ........................................... 30
第四章 討論 ..................................................................... 34
第五章 結論 ..................................................................... 39
第六章 參考文獻 .............................................................. 42
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