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

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。在这个体细胞重排过程中，组成基因片段被组装起来产生 B细胞和T细胞发育不同阶段的功能AGR基因。每个基因片段的两侧都有一个 V(D)J重组酶识别和切割的重组信号序列(RSS)，包含 RAG1和RAG2，在V(D)J重组的第一步。由于RSS只是半保守的， V(D)J重组酶必须能够在大范围的变异RSS上切割以产生不同的AGR 剧目。然而，有数百万个神秘的RSS样位点(CRs)位于整个基因组中。 错误的RAG介导的CRSS位点的切割会导致致癌的染色体重排。 因此，RAG1/2必须是混杂的，以促进AGR基因座上保守程度较低的RSS的重组，但 它还必须精确，以避免偏离目标的cRSS。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，对常规 与异常V(D)J重组事件的对比。探讨DNA序列选择性对V(D)J的贡献 重组，我们发展了一种高通量重组方法来分析RSS的选择性，其中 通过对下一代RSS基底库的分析，得到了V(D)J重组在RSS基底库上的相对效率 测序结果。使用这种方法，我们将实证地表征增强RAG1/2活性的RSS基序 形成不同的抗原受体谱系，以及确定不太理想的RSS基序 非常规的V(D)J复合反应。我们的初步研究已经产生了信息量很大的结果， 它们显示了首选的序列基序和不同区域之间的序列相互依赖关系 对V(D)J重组活性有显著影响的RSS。此外，特定的RSS 基序似乎更倾向于非常规的V(D)J复合反应。在我们初步的基础上 结果，我们假设RSS内的特定关系通过以下方式影响它们的相对利用率 RAG蛋白，2)受特定的染色质环境调节，3)在传统的 与异常的V(D)J重组反应相比。总体而言，我们预测这个项目的结果将显著 改善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 分别进行复合反应。

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.