Chromatin regulation of TCR locus V(D)J recombination

TCR 位点 V(D)J 重组的染色质调控

• 批准号: 10397568
• 负责人: Michael S Krangel
• 金额: $ 50.76万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10397568
• 关键词: Adaptive Immune System; Address; Alleles; Antigen Receptors; Antigens; Autoimmunity; B-Lymphocytes; Characteristics; Chromatin; Chromatin Structure; Chromosomal translocation; Complex; DNA; DNA Damage; Development; Ensure; Event; Exclusion; Failure; Generations; Genes; Genetic Recombination; Genetic Transcription; Immune system; Immunologic Deficiency Syndromes; Instruction; Jaw; Knowledge; Lymphocyte; Lymphocyte Subset; Malignant Neoplasms; Maps; Modification; Molecular; Molecular Conformation; Mus; Nuclear Lamina; Outcome; Pathology; Play; Production; Receptor Gene; Regulation; Role; Specificity; Systems Development; T cell regulation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; Thymocyte Selection; Tumor stage; V(D)J Recombination; Vertebrates; Work; adaptive immunity; base; combinatorial; genetic manipulation; genome integrity; genomic locus; in vivo; insight; novel; programs; receptor; recombinase; response; thymocyte; tumorigenesis

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.

工作总结 通过 V(D)J 重组进行抗原受体 (AgR) 基因的体细胞组装，产生多样化的抗原 T 和 B 淋巴细胞的受体库，是有颌脊椎动物适应性免疫的基础。 AgR 基因包含在大型且复杂的基因位点中，必须在多个水平上对其进行调节 实现早期淋巴细胞发育的重要结果。重组必须是发育性的 命令允许生产和选择具有适当特征的淋巴细胞亚群，并在 适当的数字。重组可能受到等位基因调控，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。重组必须受到保障措施的约束 确保基因组完整性。重组还必须具有足够的随机性才能生成 AgR 库 其特点是广泛的组合多样性。 RAG 重组酶活性通过以下方式靶向 AgR 位点： 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 位点的亚核区室化。 我们的研究旨在揭示支持发育调控的基本分子机制 体内胸腺细胞发育过程中 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进行重组并有助于 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周期， 最初的 Tcrd 重排，随后是一系列初级和次级 Tcra 重排， 每一个删除重排都会替换之前的一个。我们已经深入了解了如何做到这一点 重组程序是在染色质水平上执行的，现在寻求了解重组程序的两个主要方面 这个程序。首先，Tcrd重组在组合多样性的发展中发挥什么作用 Tcra 曲目？其次，DNA 损伤反应在 Tcra 的演替过程中起什么作用？ 重新安排？时间调节对于基于 TCR 表达进行胸腺细胞选择至关重要 重排的 Tcra 基因之前的蛋白质被随后的 Tcra 重组事件删除。其中 TCR 位点，仅 Tcrb 受到等位基因排除。该位点也非常不寻常，因为它与 核层 (NL)，通常被认为抑制转录和 V(D)J 的区室 重组。然而，在发育阶段，当基因座最初出现时，该基因座与 NL 相关联。 转录并进行重排。我们最近的工作提供了地图和机制的见解 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 关联 调节体内胸腺细胞发育中的 Tcrb 重排和 Tcrb 等位基因排除。我们将解决 通过生成和分析新的基因操纵小鼠品系来解决这些问题。