Elucidating the Role of NEMO and SpiC in DNA Double Strand Break Induced Inhibition of V(D)J Recombination

Abstract

Abstract V(D)J recombination is regulated such that most lymphocytes assemble and express an antigen receptor from only one allele (allelic exclusion). In pre-B cells, RAG-induced DNA double strand breaks (DSBs) at Igκ loci signal via ATM to rapidly repress Rag1/2 transcription, inhibit accessibility of Igk loci, and limit V recombination. We hypothesize that RAG DSB-induced repression of Rag1/2 is critical to transiently limit V recombination, ensure allelic exclusion, and inhibit oncogenic Ig translocations. Fittingly, Atm−/− mice have higher frequencies of developing B cells with RAG DSBs at both Igh and Igk loci and of mature B cells with bi-allelic expression of these genes. Yet, given the multifunctional roles of ATM in the DSB response, Atm−/− phenotypes cannot be directly attributed to Rag1/2 repression. To test our hypothesis, we study mouse models lacking downstream effectors of ATM: i) SpiC, which suppresses Igk accessibility in response to RAG DSBs, and ii) NFκB essential modulator (NEMO), which represses Rag1/2 in response to genotoxic DSBs. I show here that the frequency of surface bi-allelic Igκ expression is normal on SpiC−/− B cells and increased on Nemo−/− B cells. I also show that Nemo−/− pre-B cells exhibit impaired Rag1/2 repression in response to RAG DSBs and concomitant increased RAG cleavage at Igk. Our data support that RAG DSB-induced repression of Rag1/2 transiently inhibits additional Igk rearrangements to enforce Igκ allelic exclusion independent of ATM-mediated DSB repair. I am using Nemo−/− mouse models to determine if DSB-induced Rag1/2 repression requires Nemo in pro-B cells, and if this mechanism is critical to suppress oncogenic Ig translocations independent of defects in DSB repair/checkpoint activation.