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.

项目摘要 自适应免疫系统中抗原受体（AGR）的大量曲目取决于V（d）J 重组。在这个体细胞重排过程中，组件基因段被组装成生成 B和T细胞发育的不同阶段的功能性AGR基因。每个基因片段侧面 重组信号序列（RSS），由V（d）J重组酶识别并裂解，含有 RAG1和RAG2，在V（d）J重组的第一个酶促步骤中。由于RSS仅是半保守的， V（d）J重组酶必须能够在广泛的变体RSS上分裂以产生多种AGR 曲目。但是，整个基因组都有数百万个隐形的RSS样位点（CRS）。 CRSS位点上错误的RAG介导的裂解可能会导致致癌染色体重排。 因此，RAG1/2必须是混杂的，以促进AGR基因座保守不良的RSS的重组，但是 避免脱靶CRSS也必须精确。关于DNA的贡献，长期存在的问题仍然存在 序列选择性以及染色质环境的影响，对传统之间的平衡 与异常V（D）J重组事件。解决DNA序列选择性对V（d）J的贡献 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 V（d）J重组对RSS底物库的相对效率是通过分析下一代获得的 测序结果。使用此方法，我们将凭经验表征增强RAG1/2活性的RSS基序 塑造多样的抗原受体库，并识别有利于 规定的V（d）J重组反应。我们的初步研究产生了丰富的结果， 已经显示出优选的序列基序和序列之间的相互依存关系 RSS对V（d）J重组活性的水平产生重大影响。此外，特定的RSS 图案似乎优先偏爱非惯例V（d）J重组反应。基于我们的初步 结果，我们假设RSS中的特定关系1）影响其相对利用 抹布蛋白，2）由特定的染色质环境调节，3）在常规中控制其命运 与异常V（D）J重组反应。总体而言，我们预测该项目的发现将大大显着 在正常和异常V（d）J中提高我们对RSS和CRSS的破布选择性的当前理解 重组反应分别。