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研究生:張筱茹
研究生(外文):Shiao-Ru Chang
論文名稱:KAP1羧酸端結構功能研究
論文名稱(外文):Functional study of C-terminal domain on KAP1
指導教授:楊文明楊文明引用關係
指導教授(外文):Wen-Ming Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:72
中文關鍵詞:轉錄抑制聚合作用分子內交互作用
外文關鍵詞:KAP1transcriptional repressionoligomerizationintra-molecular interaction
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真核生物的基因表現主要由透過轉錄階層進行調控,其中輔因子KAP1 (KRAB-associated protein 1) 存在許多轉錄複合體中,參與轉錄抑制作用。研究指出具有KRAB domain的鋅指轉錄因子 (KRAB-ZFPs) 抑制其目標基因轉錄需透過KAP1連結調控蛋白,以抑制目標基因的轉錄。但目前對於KAP1如何扮演連結轉錄因子與調控蛋白的媒介分子仍尚未清楚。過去研究發現,KAP1透過其胺根端RBCC domain進行聚合形成多聚體,並同時以單體和多聚體形式共同存在細胞內,但不同形式的KAP1如何調控轉錄仍屬未知。因此本篇研究首先將針對KAP1如何透過其自身分子間的聚合作用影響其轉錄活性進行研究。此外,本實驗室研究指出:KAP1利用其羧酸端bromodomain與胺根端RBCC domain發生分子內交互作用,促使KAP1形成具有轉錄抑制活性之形式。綜合上述研究,推測KAP1的轉錄活性可能藉由其胺根端之RBCC domain發生分子間與分子內的交互作用進行協同調控。因此本篇論文將進一步針對KAP1如何透過分子間與分子內的交互作用,調控KAP1在細胞內的分布情形以及轉錄抑制活性進行研究。
首先,為了瞭解KAP1如何透過其自身分子間的聚合作用影響其轉錄調控活性,本篇論文利用點突變破壞KAP1自身分子間的進行聚合的胺基酸,模擬KAP1單體。結果發現KAP1單體傾向分布於細胞核內結構緊密的異染色質區域,並具有更強的轉錄抑制活性。根據以上研究結果,推論具有轉錄抑制活性的KAP1以單體形式存在細胞內。
為了進一步瞭解KAP1如何透過自身分子間的聚合作用影響KAP1在細胞內的分布及其轉錄抑制活性,本篇論文從KAP1羧酸端結構進行研究。根據本實驗室研究發現,KAP1失去其羧酸端bromodomain,造成KAP1在細胞核內結構鬆散的常染色質區形成亮點狀分布,並失去部份轉錄抑制活性。但這些由失去bromodomain造成的現象,都可透過外加羧酸端bromodomain與胺根端RBCC domain發生分子內交互作用,回復KAP1在細胞核內的分布情形及其轉錄活性。綜合上述研究,進一步推測:KAP1藉由其bromodomain與進行聚合的RBCC domain發生分子內交互作用,調控KAP1自身分子間的聚合作用,進而影響KAP1在細胞核內的分布情形及其轉錄抑制活性。為了驗證上述假設,首先本篇論文利用共同免疫沈澱法證實KAP1藉由其羧酸端bromodomain與胺根端RBCC domain發生分子內交互作用,接著為了驗證KAP1羧酸端bromodomain藉由與負責聚合的RBCC domain發生分子內交互作用以調控KAP1自身分子間的聚合作用,本篇研究利用層次比重離心法分析,證明KAP1失去其羧酸端bromodomain後傾向以多聚體形式存在,並且透過外加bromodomain增加其單聚體形式,證明KAP1分子內的交互作用影響其自身分子間的聚合作用。
本篇論文研究發現:KAP1透過其羧酸端的bromodomain與胺根端負責進行聚合的RBCC domain發生分子內的交互作用以影響其分子間的聚合,導致KAP1形成具有轉錄抑制活性的單體形式,並分布於細胞核內結構緊密的異染色質區。

Transcription regulation plays a critical role in eukaryotic gene expression. KAP1 (KRAB-associated protein) is a transcriptional intermediary factor for repression, which asts as scaffold in many transcriptional regulation complexes. KAP1 serves as a co-repressor for KRAB-containing zinc finger proteins (KRAB-ZFPs) in the regulation of gene expression and KAP1 is required for KRAB-ZFPs-mediated transcription repression. However, the molecular mechanism of KAP1 functions as a co-repressor to connect transcription factors and regulatory proteins is unclear.
Previous studies proved that N-terminal RBCC domain of KAP1 is responsible for oligomerization and KAP1 exists as both oligomers and monomers in cells, but how KAP1 oligomerization regulates transcriptional repression remains unknown. First, we want to understand wether KAP1 oligomerization affects its function on transcriptional repression. In addition, it has been previously demonstrated that C-terminal bromodomain (BD) of KAP1 regulates subcellular localization and transcriptional activity by interacting with its N-terminal RBCC domain. Above studies suggest that intramolecular and intermolecular interaction of KAP1 might cooperative regulate subcellular localization and transcriptional activity of KAP1. In this study, we want to address the molecular mechanism of intermolecular and intramolecular interaction of KAP1 regulates its subcellular localization and transcriptional activity.
To investigate the role of KAP1 oligomerization in transcriptional repression, we blocked the self-interaction of KAP1 to mimic monomeric KAP1 by generating point mutations on RBCC domain. Here we demonstrated that monomeric KAP1 exhibited a decreased level of euchromatin localization but stronger repressional activity. The results suggest that monomeric KAP1 is an active form for transcriptional repression.
Go further to explore the mechanism of KAP1 oligomerization in subcellular localization and transcriptional activity, we approach it from C-terminal domain of KAP1. Previously we found that missing of BD in KAP1 results in spots formation in euchromatin region and derepressional activity, but these phenomena could be reverted to wild type by addition of BD which through intramolecular interaction with RBCC domain. Thus, we proposed that intramolecular interaction mediated by BD of KAP1 contribute to subcellular localiztion and transcriptional activity by promoting KAP1 monomers formation to repress transcription, which blocks the RBCC domain for KAP1 oligomerization. To prove this idea, first, we perform co-immunoprecipitation assay to prove C-terminal BD interacts with N-terminal RBCC domain. To validate that BD of KAP1 regulates its oligomerization by intramolecular interaction, we demonstrated that missing BD of KAP1 leads to oligomers formationin glycerol gradient analysis, which reverted to monomers by addition of BD. Furthermore, we demonstrated that BD interacts with RBCC domain of KAP1, suggesting that intramolecular interaction mediated by BD regulates KAP1 oligomerization.
Taken together, the results demonstrate that intramolecular interaction between C-terminal BD and N-terminal RBCC domain regulates KAP1 monomers formation, which is capable of heterochromatin localization and transcriptional repression.


壹、緒論……………………………………………………………………1
一、前言………………………………………………………………1
二、真核細胞的轉錄調控機制………………………………………1
(一)真核生物的基因表現………………………………………..1
(二)真核細胞的轉錄調控………………………………………..2
1.順/反勢轉錄調控……………………………………….....2
2.染色質結構………………………………………………..3
三、KAP1的背景介紹………………………………………………...6
(一) KAP1的發現………………..……………………………....6
(二) KAP1的分子結構…………………………………………...7
(三) KAP1的轉錄調控機制……………………………………...8
1. KAP1分子間的調控機制………………………………...8
2. KAP1分子內的調控機制……………………………….12
(四) KAP1的後轉譯修飾作用………………………………….13
1. KAP1的小泛素化修飾………………………………….14
2. KAP1的磷酸化修飾…………………………………….15
四、研究目的………………………………………………………...16
五、研究策略………………………………………………………..17
貳、材料與方法…………………………………………………………..19
一、質體構築………………………………………………………..19
二、細胞培養與基因轉移感染……………………………………..22
三、免疫共同沈澱法………………………………………………..23
四、膠體電泳法及西方墨點法………………...…………………...23
五、非還原性膠體電泳法…………………………………………...25
六、層次比重離心法………………………………………………..26
七、免疫螢光染色法………………………………………………..27
八、轉錄活性檢測…………………………………………………..27
參、結果…………………………………………………………………..29
一、突變型KAP1單體不影響其自身分子間的交互作用,但可減弱其聚合形成多聚體之能力……………………………………..29
二、突變型KAP1單體在細胞內以單體形式存在…………………30
三、KAP1單體傾向分布於結構緊密的異染色質區………………31
四、KAP1單體具有較強的轉錄抑制活性…………………………31
五、KAP1羧酸端bromodomain與負責聚合的RBCC domain發生分子內交互作用…………………………………………………..32
六、KAP1羧酸端bromodomain利用分子內交互作用調控KAP1分子間的聚合………………………………………………………..33
肆、討論…………………………………………………………………..34
一、具有轉錄抑制活性之KAP1以單體形式存在…………………34
二、KAP1分子內交互用調控其分布情形與轉錄活性……………38
三、KAP1透過其分子內交互作用調控其分子間的聚合…………39
伍、參考文獻……………………………………………………………..42
陸、圖表…………………………………………………………………..48
柒、附圖…………………………………………………………………..62

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