Chromatin regulation of TCR locus V(D)J recombination

工作总结 通过V(D)J重组对抗原受体(AGR)基因进行体细胞组装，产生多样化的抗原 T和B淋巴细胞的受体，是颌骨脊椎动物适应性免疫的基础。 AGR基因包含在大而复杂的遗传位点中，必须在多个水平上进行调控，以 达到早期淋巴细胞发育的基本结果。重组必须是发展性的 允许生产和选择具有适当特征的淋巴细胞亚群 适当的数字。重组可能受到等位基因的调节，以确保每个淋巴细胞 表达具有独特抗原特异性的单一受体。重组必须受到下列保障措施的约束 确保基因组的完整性。重组还必须具有足够的随机性，以生成AGR曲目 以广泛的组合多样性为特征。RAG重组酶活性以AGR基因座为靶点 多种基于染色质的机制，包括对AGR染色质结构的局部修改，大规模 AGR基因座的构象特征、远距离DNA接触和亚核区划。 我们的研究旨在揭示支持发育调节的基本分子机制。 胸腺细胞发育过程中T细胞受体基因的V(D)J重组在各种AGR中 TCRA-TCRD基因座的复杂性是无与伦比的，因为它包含两组基因片段， 在T细胞发育的不同阶段进行重组并促进TCR链的形成 由不同的T细胞亚群表达。值得注意的是，单个等位基因可以经历多个周期的重排， 在最初的TCRD重排之后是一系列的初级和次级TCRA重排， 每次删除重排替换先前的一个重排。我们已经对这一点有了实质性的见解 重组计划是在染色质水平上强制执行的，现在试图了解两个主要方面 这个节目。首先，TCRD重组在组合多样性的发展中扮演什么角色？ TCRA曲目？第二，DNA损伤反应在TCRA继发过程中的作用是什么 重新安排？时间调节是允许基于TCR表达的胸腺细胞选择的关键 在重排的TCRA基因之前的蛋白质被随后的TCRA重组事件删除。在这些人中 TCR基因座，只有Tcrb受到等位基因排斥。这个基因座也非常不寻常，因为它与 核层(NL)，通常被认为是对转录和V(D)J的抑制 重组。然而，在发育阶段，该基因座最初是与内侧线相联系的 被转录并经历了重排。我们最近的工作既提供了地图，也提供了对 TCRB-NL相互作用。我们现在计划利用这一知识来确定如何与NL建立联系 在体内调节胸腺细胞发育中的Tcrb重排和Tcrb等位基因排斥。我们将解决 这些问题是通过产生和分析新的基因操纵小鼠品系来实现的。

Summary of Work The somatic assembly of antigen receptor (AgR) genes by V(D)J recombination creates the diverse antigen receptor repertoires of T and B lymphocytes and is fundamental to adaptive immunity in jawed vertebrates. AgR genes are contained within large and complex genetic loci that must be regulated at multiple levels to achieve essential outcomes of early lymphocyte development. Recombination must be developmentally ordered to allow the production and selection of lymphocyte subsets with appropriate characteristics and in appropriate numbers. Recombination may be subject to allelic regulation to ensure that each lymphocyte expresses a single receptor with unique antigen specificity. Recombination must be subject to safeguards that ensure genomic integrity. Recombination must also be sufficiently stochastic to generate AgR repertoires characterized by extensive combinatorial diversity. RAG recombinase activity is targeted to AgR loci by multiple chromatin-based mechanisms, including local modifications to AgR chromatin structure, large-scale conformational features and long-distance DNA contacts, and subnuclear compartmentalization of AgR loci. Our studies aim to reveal fundamental molecular mechanisms underpinning the developmental regulation of V(D)J recombination at T cell receptor (TCR) loci in developing thymocytes in vivo. Among the various AgR loci, the Tcra-Tcrd locus is unrivaled in its complexity, because it contains two sets of gene segments that undergo recombination at different stages of T cell development and contribute to the formation of TCR chains expressed by distinct subsets of T cells. Notably, a single allele can undergo multiple cycles of rearrangement, with initial Tcrd rearrangement followed by a succession of primary and secondary Tcra rearrangements, with each deletional rearrangement replacing the one prior. We have developed substantial insights into how this recombination program is enforced at the level of chromatin, and now seek to understand two major aspects of this program. First, what role does Tcrd recombination play in the development of a combinatorially diverse Tcra repertoire? Second, what is the role of the DNA damage response in pacing the succession of Tcra rearrangements? Temporal regulation is essential to allow thymocyte selection based on expression of TCR proteins before the rearranged Tcra gene is deleted by a subsequent Tcra recombination event. Among the TCR loci, only Tcrb is subject to allelic exclusion. This locus is also highly unusual in that it associates with the nuclear lamina (NL), a compartment generally considered repressive for transcription and V(D)J recombination. Yet the locus associates with the NL during the developmental stage when the locus is initially transcribed and undergoes rearrangement. Our recent work provides both a map and mechanistic insight into Tcrb-NL interactions. We now plan to leverage this knowledge to determine how association with the NL regulates Tcrb rearrangement and Tcrb allelic exclusion in developing thymocytes in vivo. We will address these questions by generation and analysis of novel strains of genetically manipulated mice.