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 适应性免疫系统中大量的抗原受体（AgR）依赖于V（D）J 重组在这种体细胞重排过程中，组分基因片段被组装以产生 功能性AgR基因在B和T细胞发育的不同阶段。每个基因片段的侧翼都有一个 重组信号序列（RSS），其被V（D）J重组酶识别和切割，包含 RAG 1和RAG 2，在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重组酶必须能够在宽范围变体RSS处切割以产生不同的AgR 保留曲目然而，在整个基因组中有数百万个隐藏的RSS样位点（cRSS）。 错误的RAG介导的cRSS位点切割可导致致癌染色体重排。 因此，RAG 1/2必须是混杂的，以促进AgR基因座上保守性差的RSS的重组， 它还必须精确以避免偏离目标cRSS。关于DNA的贡献， 序列选择性，沿着染色质环境的影响，在常规的 相对于异常V（D）J重组事件。为了解决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 通过对下一代的分析，获得了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，我们将经验性地表征增强RAG 1/2活性的RSS基序 形成一个多样化的抗原受体库，以及确定次优的RSS基序，有利于 非常规V（D）J复合反应。我们的初步研究已经产生了非常丰富的结果， 其显示了优选的序列基序和不同区域之间的序列相互依赖性， RSS对V（D）J重组活性水平具有显著影响。具体RSS 基序似乎优先有利于非常规的V（D）J复合反应。根据我们初步的 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）由特定的染色质环境调节，3）在常规染色质环境中控制它们的命运。 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 RAG选择性的理解 分别进行重组反应。