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研究生:李慧真
研究生(外文):Huei-Jhen Li
論文名稱:T細胞接受器Jα基因座之DNA結構及組蛋白修飾對V(D)J重組之調控
論文名稱(外文):DNA conformation and histone modifications in the T-cell receptor Jα locus coordinate Vα-to-Jα recombination
指導教授:果伽蘭
口試委員:王惠鈞余榮熾孔祥智張瀞仁
口試日期:2016-07-25
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
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:2016
畢業學年度:104
語文別:中文
論文頁數:98
中文關鍵詞:T細胞T細胞接受器T細胞接受器α鏈V(D)J基因重組免疫反應
外文關鍵詞:T cellT cell receptorT cell receptorαchainV(D)J recombinationImmune response
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淋巴細胞利用重組活化基因 (recombination activating gene, RAG1 and RAG2)產物,使V或D及J基因片段與重組訊息序列(recombination signal sequences, RSS)間產生斷裂,再由non-homologous end-joining修復作用使V或D及J基因片段結合,即所謂的V(D)J recombination。又accessibility理論假設RAG1及RAG2僅於細胞分化之特定時點可以結合特定基因片段之RSS,使V(D)J recombination過程具細胞發展階段專一性及重組位置專一性之特徵。然而特別是在wild type重組活化的染色質中,重組過程的分子調控機制則仍待實驗研究。本論文藉由探討native Jα 基因座(locus),發現T細胞接受器(T cell receptor, TCR) α鏈的Vα與Jα基因重組過程,係受全面性與局部性之調控。全面性調控為在不同分化階段的胸腺細胞中,藉由組蛋白修飾(histone modification),如histone H3及H4的acetylation (H3Ac、H4Ac)、H3K4的trimethylation (H3K4me3)及H3K9的dimethylation (H3K9me2),影響Jα基因座結構的鬆散程度並調控RAG1及RAG2結合Jα基因座的能力及數量。另本論文研究發現,在in vivo的RAG1及RAG2結合Jα基因座時,並非僅限於RSS序列上,表示尚有其他因素調控此具RSS專一性的DNA裂解反應,即局部性之調控。研究結果顯示,在in vitro的CD實驗所偵測之DNA結構,與in vivo已知的Jα基因片段使用機率間,具高度關聯性,而且影響基因重組的DNA結構為RSS及Jα整體的DNA序列,而非僅有RSS或Jα的DNA序列所造成。
綜上,影響wild type Jα基因座進行V(D)J recombination的因素包括全面性的整體染色質的鬆散程度與可獲得性,及局部RSSJα的DNA結構可被RAG1結合並進行裂解反應的程度。本論文可作為後續探討DNA結構如何影響RAG結合專一性之方向,並可應用於研究臨床上常見的白血病或不正常的gene translocation現象所產生疾病的致病機轉。

Lymphocytes use recombination activating gene products (RAG1 and RAG2, referred to as V(D)J recombinase) to cleave recombination signal sequences (RSS) that flank V, D and J gene segments. Non-homologous end-joining repair follows and results in V(D)J gene assembly. The accessibility theory proposes that a RSS is accessible to V(D)J recombinase only at specific times, which accounts for how V(D)J rearrangement occurs in a developmental stage- and site-specific manner. However, precise molecular mechanism of regulation remains unclear, especially in the context of native, recombination-active chromatin. Here we demonstrate that at the native Jα locus, T-cell receptor (TCR) Vα to Jα gene segment recombination is regulated both globally and locally. Globally, developmental stage-specific histone-modifications (acetylations, H3K4me3 and H3K9me2) cooperatively regulate Jα locus-accessibility to V(D)J recombinase. Strikingly, recombinase-binding at the Jα locus is not restricted to RSS, indicating beyond locus-accessibility regulation for RSS-specific DNA-cleavage in vivo. Locally, we identified an excellent correlation between DNA conformation detected by circular dichroism (CD) in vitro and Jα gene recombination-competency in vivo. Such conformation-recombination correlation exists in DNA block composed of RSS and its cognate Jα, not in separate RSS or Jα. Together, we propose that global locus-accessibility and local RSS-encompassing DNA sequence/conformation coordinate Vα-to-Jα recombination at the native Jα locus. We anticipate our findings to be a starting point for studies of how DNA structure directs V(D)J recombinase substrate-specificity. Such knowledge is of clinical interest because it will contribute to define the illegitimate chromosomal translocation, causative of leukemia and involving cryptic site-targeting by V(D)J recombinase.

中文摘要 i
Abstract iii
目錄 v
縮寫表 viii
圖目錄 ix
表目錄 xi
第一章 前言 1
第一節 免疫系統 1
第二節 B細胞及T細胞 2
第三節 T細胞接受器 3
第四節 T細胞接受器基因座 3
第五節 T細胞接受器基因重組 5
第六節T細胞分化 8
第七節 組蛋白修飾 10
第八節 RSS序列分析工具 11
第九節 本論文主要探討議題 11
第二章 實驗材料與方法 15
第一節 實驗材料 15
第一項 胸腺 15
第二項 周邊血液淋巴球 15
第二節 方法 15
第一項 分離胸腺細胞 16
第二項 分離PBL 16
第三項 CD4、CD8、DN及DP細胞分離 17
第四項 選殖(Cloning) 17
第五項 DNA定序 19
第六項 染色質免疫沈澱反應 20
第七項 即時聚合酶鏈反應(real-time PCR) 20
第八項 Weight models 21
第九項 RIC數值計算 22
第十項 Circular Dichroism Spectroscopy (CD) 22
第三章 結果與討論 23
第一節 Jα基因座之組蛋白修飾 23
第一項 Histone acetylation 24
第二項 Histone H3K4 trimethylation 25
第三項 Histone H3K9 dimethylation 26
第二節 Jα基因座之RAG結合情形 27
第三節 Jα基因座序列專一性重組之調控 30
第一項 DNA序列分析 30
第二項 DNA結構分析 32
第四章 結論及未來研究 35
參考文獻 92
附錄 97

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