DNA sequence selectivity in conventional and aberrant V(D)J recombination

常规和异常 V(D)J 重组中的 DNA 序列选择性

• 批准号: 10586433
• 负责人: Karla K Rodgers
• 金额: $ 45.15万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-07 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10586433
• 关键词: Adaptive Immune System; Address; Antigen Receptors; B-Lymphocytes; Base Pairing; Binding; Binding Proteins; Biochemical; Biological Assay; Cells; Chromatin; Chromosomal Rearrangement; Complex; Coupled; DNA; DNA Double Strand Break; DNA Sequence; Development; Disease; Elements; Environment; Ephrin-A5; Equilibrium; Event; Gene Proteins; Gene Rearrangement; Genes; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; High-Throughput Nucleotide Sequencing; Histone H3; IgK; In Vitro; Libraries; Lymphocyte; Mediating; Methods; Mus; Mutate; Nonhomologous DNA End Joining; Oncogenic; Outcome; Pattern; Peptide Signal Sequences; Process; Rag1 Mouse; Reaction; Receptor Gene; Risk; Shapes; Signal Transduction; Site; System; T-Cell Development; T-Lymphocyte; Testing; V(D)J Recombination; Variant; base; defined contribution; experimental study; improved; insight; mouse model; mutant; next generation sequencing; promoter; recombinase

Project Summary The vast repertoires of antigen receptors (AgRs) in the adaptive immune system are dependent on V(D)J recombination. In this somatic rearrangement process, component gene segments are assembled to generate functional AgR genes during distinct stages of B and T cell development. Each gene segment is flanked by a recombination signal sequence (RSS) which is recognized and cleaved by the V(D)J recombinase, containing RAG1 and RAG2, in the first enzymatic steps of V(D)J recombination. As the RSSs are only semi-conserved, the V(D)J recombinase must be capable of cleaving at a wide range of variant RSSs to generate diverse AgR repertoires. However, there are millions of cryptic RSS-like sites (cRSS) that are located throughout the genome. Erroneous RAG-mediated cleavage at cRSS sites can cause oncogenic chromosomal rearrangements. Therefore, RAG1/2 must be promiscuous to facilitate recombination of poorly conserved RSSs at AgR loci, but it must also be precise to avoid off-target cRSSs. Long standing questions remain as to the contribution of DNA sequence selectivity, along with the effects of the chromatin environment, on the balance between conventional versus aberrant V(D)J recombination events. To address the contribution of DNA sequence selectivity to V(D)J recombination, we developed a high-throughput recombination method to analyze RSS selectivity, in which the relative efficiency of V(D)J recombination on RSS substrate libraries are obtained by analysis of next generation sequencing results. Using this method, we will empirically characterize RSS motifs that enhance RAG1/2 activity to shape a diverse antigen receptor repertoire, as well as identify suboptimal RSS motifs that favor nonconventional V(D)J recombination reactions. Our preliminary studies have yielded highly informative results, which have shown preferred sequence motifs and sequence interdependencies between different regions of the RSS that have significant consequences on the level of V(D)J recombination activity. Furthermore, specific RSS motifs appear to preferentially favor nonconventional V(D)J recombination reactions. Building on our preliminary results, we hypothesize that the specific relationships within RSSs 1) influence their relative utilization by the RAG proteins, 2) are modulated by specific chromatin environments, and 3) govern their fate in conventional versus aberrant V(D)J recombination reactions. Overall, we predict that findings from this project will significantly improve our current understanding of RAG selectivity of RSSs and cRSSs in normal and aberrant V(D)J recombination reactions, respectively.

项目概要 适应性免疫系统中大量的抗原受体 (AgR) 依赖于 V(D)J 重组。在这个体细胞重排过程中，组成基因片段被组装以产生 在 B 细胞和 T 细胞发育的不同阶段发挥功能的 AgR 基因。每个基因片段的两侧都有一个 重组信号序列 (RSS)，被 V(D)J 重组酶识别和切割，包含 RAG1 和 RAG2，处于 V(D)J 重组的第一个酶促步骤中。由于 RSS 只是半保守的， V(D)J 重组酶必须能够切割各种不同的 RSS 变体以产生不同的 AgR 剧目。然而，整个基因组中存在数百万个神秘的类 RSS 站点 (cRSS)。 cRSS 位点上 RAG 介导的错误切割可导致致癌染色体重排。 因此，RAG1/2 必须是混杂的，以促进 AgR 位点处保守性差的 RSS 的重组，但是 它还必须精确以避免偏离目标的 cRSS。关于 DNA 贡献的长期疑问仍然存在 序列选择性以及染色质环境的影响对常规之间的平衡 与异常 V(D)J 重组事件相比。解决 DNA 序列选择性对 V(D)J 的影响 重组，我们开发了一种高通量重组方法来分析 RSS 选择性，其中 通过下一代分析获得 V(D)J 重组在 RSS 底物库上的相对效率 测序结果。使用这种方法，我们将凭经验表征增强 RAG1/2 活性的 RSS 基序 形成多样化的抗原受体库，并识别有利于的次优 RSS 基序 非常规 V(D)J 重组反应。我们的初步研究取得了信息丰富的结果， 已显示出不同区域之间的优选序列基序和序列相互依赖性 RSS 对 V(D)J 重组活性水平有显着影响。此外，具体的RSS 基序似乎优先支持非常规 V(D)J 重组反应。在我们初步的基础上 结果，我们假设 RSS 中的特定关系 1) 影响它们的相对利用率 RAG 蛋白，2) 受特定染色质环境调节，3) 控制它们在常规中的命运 与异常的 V(D)J 重组反应相比。总的来说，我们预测该项目的发现将显着 提高我们目前对正常和异常 V(D)J 中 RSS 和 cRSS 的 RAG 选择性的理解 分别为重组反应。