E2A/HEB TRANSCRIPTION FACTORS IN T CELL DEVELOPMENT

通过V(D)J重组的T细胞受体（TCR）基因重排为适应性免疫提供了结构基础。这种基因重排事件是淋巴系统所特有的，并产生丰富的T细胞库，这些T细胞能够在免疫应答过程中识别多种肽抗原。在胸腺T细胞发育过程中，控制单个TCR基因转录的精确时间和选择性的调控分子知之甚少。最近的研究表明，转录因子E2A和HEB在启动淋巴细胞特异性V(D)J重组事件中起重要作用。在我们自己的实验室进行的遗传学研究进一步表明，E2A/HEB直接参与了TCR β位点的基因重排。在本提案中，我们计划专门研究E2A/HEB在T细胞发育过程中TCR β基因转录和重排中的作用。首先，我们假设E2A和HEB转录因子直接参与了TCR V β基因的转录，以确保所有V基因都有机会被用于重排。其次，这种E2A/HEB活性必须在DP阶段下调，以防止TCR β基因的双等位基因表达，这一过程被称为等位基因排斥。我们已经开发了几种小鼠模型，允许对E2A/HEB在T细胞发育中的功能进行遗传和生化研究。特别是，表达标记E2A分子的E2A敲入小鼠将用于染色质免疫沉淀试验，以确定E2A在TCR β位点中的位置和时间。基因敲除小鼠将用于确定E2A/HEB和TCR基因表达之间的因果关系。虽然本研究的重点是对TCR β基因位点的解剖，但其方法和概念一般适用于理解T细胞中鉴定的其他E2A/HEB靶基因。大多数E2A/HEB靶点，包括TCR β、preT α和TCR α基因，必须协调表达以确保T细胞发育的正常进展和完成。因此，我们还将开展一项基于基因组的研究，以扩大我们对T细胞发育过程中协调基因调控事件的理解。

T cell receptor (TCR) gene rearrangement through V(D)J recombination provides the structural basis for adaptive immunity. This gene rearrangement event is unique to the lymphoid system and yields a rich repertoire of T cells which are capable of recognizing a diverse array of peptide antigens during immune responses. Very little is known about the regulatory molecules that control the precise timing and selectivity of transcription of individual TCR genes during T cell development in the thymus. Recent works have shown that the transcription factors E2A and HEB play important roles in initiating lymphoid specific V(D)J recombination events. Genetic studies carried out in our own laboratory further suggested that E2A/HEB are directly involved in gene rearrangement at the TCR beta locus. In this proposal, we plan to specifically investigate the role of E2A/HEB in TCR beta gene transcription and rearrangement during T cell development. First, we hypothesize that E2A and HEB transcription factors are directly involved in TCR V beta gene transcription to ensure that all the V genes have a chance to be used in rearrangement. Second, this E2A/HEB activity must be down regulated subsequently at the DP stage to prevent biallelic expression of the TCR beta gene, a process known as allelic exclusion. We have developed several mouse models to allow both genetic and biochemical investigations of E2A/HEB function in T cell development. In particular, an E2A knockin mouse expressing tagged E2A molecules will be used in chromatin immunoprecipitation assays to determine where and when E2A functions in the TCR beta locus. Gene knockout mice will be used to determine the causal link between E2A/HEB and TCR gene expression. While the proposed research focuses on the dissection of TCR beta gene locus, the methods and concepts are generally applicable to understanding other E2A/HEB target genes identified in T cells. Most E2A/HEB targets, including TCR beta, preT alpha, and TCR alpha genes, must be coordinately expressed to ensure the proper progression and completion of T cell development. Thus, we will also conduct a genome-based study to broaden our understanding of the coordinate gene regulation events during T cell development.