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

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.