調控T細胞接受器alpha及delta基因座內 V(D)J重組機制之組蛋白修飾與相關因子

Abstract

在T細胞表現T細胞接受器alpha/beta以及gamma/delta基因時，必須進行具有基因傳承，發展和位置特定性的V（D）J重組，而成熟的alpha/beta T細胞必須在細胞表面表現功能性的alpha/beta T細胞接受器，在整個T細胞發育的過程當中，前趨物經由骨髓進入胸腺，其依序尚未成熟的CD4-CD8-皆未表現T細胞(CD4-CD8- double negative, DN細胞)發展成CD4+CD8+皆表現的T細胞(CD4-CD8- double positive, DP細胞)，最後進入單一表現CD4+或CD8+的成熟T細胞(CD4- or CD8- single positive, SP細胞)。 V（D）J重組機制中涉及recombination activating gene products: RAG1 and RAG2重組蛋白酶的參與，其認知位於V基因、D基因和J基因片段的兩側當中具有7個和9個核苷酸保守序列的重組信號序列(recombination signal sequence, RSS ) 基因位置，除此之外，組蛋白修飾和參與T細胞發育過程中的因子，如何參與不同階段的V（D）J重組初始作用以及調控其是否進行轉錄，目前尚未有完整清楚的分子調控機制，特別是在老鼠模型中，V（D）J重組是如何進行並受調控是值得探討的方向。 而我們的研究目標主要針對C57BL/6老鼠體內的胸腺組織，進行整個T細胞接受器在V alpha 到J alpha基因序列重組過程中，組蛋白修飾作用、RAG1/2重組蛋白酶結合情形以及分子調控的相關因子研究，將T細胞分出DN及DP細胞，利用染色質免疫沉澱及定量PCR檢測不同時期T細胞進行的 V（D）J重組，驗證組蛋白修飾，包括組蛋白H3及H4乙烯化、H3K4及H3K9的不同甲基化程度及RAG1及RAG2重組蛋白酶在T細胞接受器alpha和delta基因位置中，如何調控其進行重組作用，另一實驗則利用液相層析串聯式質譜儀比對結合二維電泳蛋白質點，比較不同T細胞發育階段調控V（D）J重組時蛋白質因子的差異性。 根據染色質免疫沉澱及定量PCR的結果證實組蛋白H3甲基化在T細胞接受器V alpha至J alpha重組過程中扮演的調控角色，而我們的實驗對於重組中心位置的存在以及受到組蛋白修飾等調控，提供與RAG蛋白缺陷的老鼠模型強而有力的一致性結果，另外針對DN與DP細胞核內蛋白，藉由二維電泳和液相層析串聯式質譜儀分析，找出許多差異表現的蛋白質並比對出與V（D）J重組相關的調控因子，我們預期進一步研究這些相關蛋白質，將可能獲得許多V（D）J重組體內機制的重要分子資訊。

Lineage-, developmental- and site-specific V(D)J recombination is required for expressing T-cell receptor (TCR)αβ or γδ genes in T lymphocytes. Mature αβ T lymphocytes, defined by expressing functional αβ TCR on the cell surface, are developed in the thymus from precursor cells that pass sequentially through CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP) and CD4+CD8-/CD4-CD8+ single positive (SP) stages. Immature thymocytes can only develop into DP stage, during which TCRα gene recombination occurs, after functional production of TCRβ proteins at the DN stage. Chromatin accessibility to the lymphoid specific recombination activating (RAG1 and RAG2 )gene products (the recombinase) that bind to the recombination signal sequence flanking V, D, and J gene segments has been proposed to be the mechanism underlying this tightly regulated process. However, the precise molecular mechanism remains unclear, especially in the context of wild-type,recombination-active chromatins. The aim of this study is to decipher the factors regulating TCR Vα-to-Jα recombination in wild-type thymocytes. Two approaches were taken; one is to study the epigenetic modifications on histones and RAG1/2-bindings at the TCRα/δ locus in wild-type thymocytes, the other is to search for differentially expressed proteins in DN and DP thymocytes. The first approach would provide information on how epigenetic modifications on chromatins regulate the accessibility to the recombinase. The second approach would provide spectrums of proteins/post-translational modifications potentially involved in regulating the TCRα gene recombination. Results of chromatin immunoprecipitation (ChIP) assays identify the previous unknown roles of histone H3 methylations to the regulation of TCR Vα-to-Jα recombination. Interestingly, our ChIP data provide solid evidence to support the presence of recombination center as one of the many levels in regulating V(D)J recombination. We also identified differentially expressed proteins factors by the 2D gel electrophoresis and 1D LC-MS-MS analysis. We expect further detail analyses on these proteins would provide important molecular information on V(D)J recombination regulation in vivo.