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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  瓦登伯格氏症候群中突變型 PAX3 改變染色質結構之探討

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/151742

標題: 瓦登伯格氏症候群中突變型 PAX3 改變染色質結構之探討
The role of PAX3 mutant on chromatin changing in Waardenburg syndrome
作者: 郭依萍
Kuo, Yi-Ping
Contributors: 楊文明
Wen-Ming Yang
分子生物學研究所
關鍵字: 染色質結構;瓦登伯格氏症候群
PAX3;Waardenburg syndorme;chromatin structure
日期: 2012
Issue Date: 2013-11-07 12:20:18 (UTC+8)
Publisher: 分子生物學研究所
摘要: PAX3 扮演一個轉錄調控因子的角色,藉由調控許多與分化相關基因的表現,進而影響脊椎動物的胚胎發育,PAX3 突變會導致分化上的疾病 Waardenburg syndrome 產生。 在 Waardenburg syndrome 的研究中發現 Pax3 有兩種型式的突變方式:單一胺基酸突變 (Missense mutation) 及截斷式突變 (truncating mutation) 皆會導致疾病產生,並且發現在 Pax3 HD domain 之後的突變絕大部分都為 truncated-form mutant,而過去對於 truncating mutation 的功能及對細胞造成的影響卻未知。因此,本論文將進一步探討 truncated-form Pax3 的功能及對細胞所造成的影響。
本論文中我們將野生型 Pax3 蛋白進行突變,模擬病人體內發現的突變型 Pax3:Pax3(Q282X)、Pax3(Q313X) 及 Pax3(L396X),送入細胞中表現,結果發現 Pax3(Q282X) 具有強烈改變細胞核內染色質分佈的能力,形成 DAPI-dots;而隨著 truncated-form mutant Pax3 發生的位置越後面,包含越多 Pax3CTD 區域,改變染色質結構能力隨之減弱。從過去研究已知 WS 病人體內同時存在有野生型及突變型 Pax3,意味著兩者可能有相互影響的能力,結果顯示野生型 Pax3 會使得突變型 Pax3 會相互影響彼此在細胞內的分佈及改變染色質結構的能力。更進一步發現了 Pax3PDHD 為 Pax3 改變染色質結構的最小功能性區域,其所誘導形成的 DAPI-dots 會與異染色質標幟相互重疊;並且與真染色質標幟部分重疊,顯示此 DAPI-dots 為緊緻的異染色質區域。並且利用生化實驗法也證實了 Pax3PDHD 具有減低染色質親和性的能力。
為了了解 Pax3PDHD 改變染色質結構的機制,首先探討其兩個功能性區域,具有與 DNA 結合的能力對於其改變染色質結構是否重要,結果顯示 HD domain 及 PD domain 對於 Pax3PDHD 改變染色質結構都是非常重要的。此外,實驗結果也發現 Pax3PDHD 可能具有非專一性結合 DNA 的能力。接著我們推測 Pax3PDHD 可能需要藉由其他蛋白的幫助,進而改變染色質結構,實驗結果證實 Pax3PDHD 可與 PARP1 交互作用,但抑制細胞內 PARP1 蛋白表現,卻不影響 Pax3PDHD 改變染色質結構,顯示 PARP1 對於 Pax3PDHD 改變染色質結構是非必須的。
實驗結果發現,Pax3(Q282X) 具有改變染色質結構的能力,暗示著可能因此而導致疾病發生,因此猜想是否可藉由使 Pax3(Q282X) 失去改變染色質結構的能力,提供一治療的可能潛力。探討是否可藉由加回 Pax3(Q282X) 所失去的 Pax3CTD,使 Pax3(Q282X) 失去其改變染色質結構的能力,進而提供未來治療 Waardenburg syndrome 的可能性,結果顯示加回 Pax3CTD 的確會使 Pax3PDHD 及 Pax3(Q282X) 失去改變染色質結構的能力。
總結本論文研究結果顯示,模擬 Waardenburg syndrome mutant Pax3 證實了 Pax3(Q282X) 具有改變染色質結構的能力,使染色質在核內呈現點狀分佈,形成一個轉錄不活化的區域。
PAX3, a transcription factor, regulates many development-related gene expression to affect vertebrate development. When PAX3 protein is mutated, a differentiation disease, called Waardenburg syndrome happen. From previous study in Waardenburg syndrome, researchers has found that two kind of mutant Pax3 will cause disease, one is missense mutation, the other is truncating mutation. Previous study of PAX3 mutant mostly focus on missense mutation. The detail mechanism of how truncated mutant Pax3 leads to Waardenburg syndrome remains unclear. In this thesis we discover that Pax3(Q282X) has chromatin changing ability, through mimicry mutant Pax3 happen in Waardenburg syndrom patient. This suggests that truncated-form Pax3 might use its chromatin-changing ability to affect occurrence of Waardenburg syndrome. Therefore, in this thesis we try to investigate the role of truncated-form mutant Pax3 in Waardenburg syndrome.
To explore the role of truncated mutant Pax3 in Waardenburg syndrome, we use site-directed mutagenesis to generate Pax3 mutants which has been reported in WS patient. The result shows that Pax3(Q282X) has chromatin changing ability, but other truncated-mutants having loger Pax3CTD region have decreased chromatin changing ability. This suggets that Pax3CTD might play an important role in truncated-form mutant Pax3 in changing chromatin structure. In Waardenburg syndrome Pax3 is single allele mutant, in that a wild type Pax3 and mutant Pax3 exist in WS patient’s cell. We further mimic real condition in WS patient by investigating whether wild Pax3 and mutant can affect each other. Results show that wild type can disrupt mutant Pax3 chromatin change ability; mutant Pax3 can makes wild type Pax3 having chromatin changing ability. Further, we mapped the functional domain for mutant to Pax3 change chromatin structure and found that Pax3PDHD possesses chromatin changing ability that forms DAPI-dots. To characterize Pax3PDHD induced-DAPI dots, we stain various chromatin markers. Results show that Pax3PDHD induced-DAPI dots can associated with heterochromatin marker: HP1α、HP1β、 HP1γ、H3K9me3 and H3K27me2; and partially associated with euchromatin marker H3K4me3, implying that Pax3PDHD-induced DAPI dots associates with repressive status chromatin. To investigate whether Pax3(Q282X) and Pax3PDHD possesses the ability to compact chromatin structure, we demonstrate that Pax3(Q282X) and Pax3PDHD can decrease the accessibility of chromatin by MNase digestion assay.
To explore the mechanism of Pax3PDHD in altering chromatin structure, we demonstrate that DNA binding activity of PD and HD domain is necessary for Pax3PDHD change chromatin structure and HD domain is also important for Pax3PDHD to form protein dots. And we further use ChIP assay shows that Pax3PDHD might use non-specific DNA binding ability to change chromatin structure. On the other hand, we proposed that Pax3PDHD might through interact with histone modifier to change chromatin structure, and we find out PARP1 may be a candidate histone modifier. Although experiment results show that Pax3PDHD can interact with PARP1, however knockdown PARP1 did not disrupt Pax3PDHD’s chromatin changing ability. This suggests PARP1 might not required for Pax3PDHD to change chromatin structure.
My thesis shows that Pax3(Q282X) and Pax3PDHD can change chromatin structure and suggests that truncated-form Pax3 might use its chromatin-changing ability to affect occurrence of Waardenburg syndrome. We envisage that by disrupting Pax3(Q282X)’s chromatin changing ability could provide a potential therapeutic, and result shows that Pax3CTD can disrupts Pax3(Q282X)’s chromatin changing ability.
Taken together, the results demonstrate that truncate-form Pax3 in Waardenburg syndrome can change chromatin structure create a repressive status chromatin.
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