| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 101 === 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.
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