Chromatin regulation of TCR locus V(D)J recombination

工作概要 抗原受体（AgR）基因通过V（D）J重组进行体细胞组装，产生了多种抗原 T和B淋巴细胞的受体库，是有颌脊椎动物获得性免疫的基础。 AgR基因包含在大而复杂的遗传基因座中，这些基因座必须在多个水平上进行调控， 实现早期淋巴细胞发育的基本结果。进化必须是进化的。 为了允许产生和选择具有适当特征的淋巴细胞亚群， 适当的数字。重组可能会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。豁免必须受到保障措施的约束， 确保基因组的完整性。扩增还必须具有足够的随机性以产生AgR库 以广泛的组合多样性为特征。RAG重组酶活性通过以下方式靶向AgR基因座： 多种基于染色质的机制，包括对AgR染色质结构的局部修饰，大规模 构象特征和长距离DNA接触，以及AgR基因座的亚核区室化。 我们的研究旨在揭示基础的分子机制，支持发育调控， 体内发育中胸腺细胞T细胞受体（TCR）位点的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经历重组并有助于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重排循环， 具有初始Tcrd重排，随后是一系列初级和次级Tcra重排， 每一次缺失重排都取代之前的一次。我们已经深入了解了 重组程序是在染色质水平上实施的，现在试图了解两个主要方面， 这个戒酒会这首先，Tcrd重组在组合多样性的发展中发挥了什么作用， TCRA曲目？第二，DNA损伤反应在Tcra演替中的作用是什么？ 重新安排时间调节对于允许基于TCR表达的胸腺细胞选择至关重要 在重排的Tcra基因被随后的Tcra重组事件删除之前，重组的Tcra蛋白质被破坏。中 在TCR基因座中，只有Tcrb受到等位基因排斥。这个基因座也是非常不寻常的，因为它与 核纤层（NL），通常被认为是抑制转录和V（D）J的隔室 重组然而，在发育阶段，当基因座最初被激活时，基因座与NL相关。 转录并经历重排。我们最近的工作提供了一个地图和机械的见解， Tcrb-NL相互作用。我们现在计划利用这些知识来确定如何与NL联系 调节体内发育中的胸腺细胞中的Tcrb重排和Tcrb等位基因排斥。我们将解决 这些问题的产生和分析的新品系的基因操纵小鼠。

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.