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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42039
標題: | TIF1beta對細胞週期調控之功能分析 The Functional Analysis of TIF1beta on the Regulation of Cell Cycle |
作者: | Yu-Sheng Lin 林祐生 |
指導教授: | 呂勝春 |
關鍵字: | 磷酸化,轉錄調控, TIF1beta,HP-1,Epigenetics,Phosphorylation,Transcriptional Regulation, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | TIF1b 是一個上位遺傳調控因子,藉由與其它的染色質蛋白互相作用,能夠在基因體的特定區位上進行染色質的結構重整與轉錄調控。一般認為TIF1b 的抑制功能主要經由其與異染色質蛋白一號(Heterochromatin Protein 1, HP-1)的交互作用所達成。在TIF1b 上的HP-1 box 是主要負責這個交互作用的區段。然而,背後的調控機制仍未完全明瞭。在這篇論文中,我們建立了一個基因置換系統能夠將
內生性的蛋白質抑制,以外生性的突變蛋白質補回,藉以研究特定胺基酸位點對其功能之影響。此外,利用抗體的專一性,我們也發現TIF1b 能夠在HP-1 box 中的Ser473 上被磷酸化,而這樣的磷酸化是會隨著細胞週期而改變的。此外,利用染色質沉澱的技術,我們也觀察到這樣的磷酸化能夠確實影響到特定基因體區位上的HP-1 召集。最後,我們也觀察到了TIF1b 能夠與組蛋白乙烯基轉化脢PCAF形成錯合物,而此觀察也提供了TIF1b 做為活化因子的可能性。 The transcriptional intermediary factor 1β (TIF1β)/KRIP-1/KAP-1/TRIM28 is an epigenetic regulator correlated to transcriptional regulation and chromatin remodeling at designated genomic loci through the interaction with other chromatinic proteins. The repressive capability of TIF1β is thought to be mediated in part by its interaction with HP1. The HP1-box, PXVXL, of TIF1β is reported to be responsible for this interaction. However, the underlying regulation is still poorly understood. Here we demonstrate the construction of a gene replacement system which replaces the endogenous TIF1β with ectopically expressed ones carrying desired alterations. Also, using phospho-specific antibody, it is shown that TIF1β is phosphorylated at Ser473, which is located in the HP1-interacting domain, and the global level of this modification fluctuates with the progression of cell cycle. Furthermore, in our ChIP assay, it is shown that the overexpression of wild type and S473A but not the phospho-mimicry S473E mutant of TIF1β can preferentially enrich the recruitment of HP1β to endogenous promoter region of CDC2 and CDC25a. In conclusion, the phosphorylation of TIF1β Ser473 is demonstrated to be functionally correlated to the recruitment of HP1 protein to specific chromatinic loci. Finally, we’ve also provided the evidence that TIF1β can potentially complex with the histone acetyltransferases PCAF, and this interaction is possibly involved in the TIF1β-mediated transcriptional activation reported previously. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42039 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子醫學研究所 |
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