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

Abstract

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.