DNA DAMAGE INDUCED DURING ANTIGEN RECEPTOR GENE ASSEMBLY SIGNALS TO REGULATE MECHANISMS OF V(D)J RECOMBINATION: IMPLICATIONS FOR AUTOIMMUNITY AND GENOMIC INTEGRITY

The adaptive immune system relies on the assembly of unique antigen receptor (AgR) genes through V(D)J recombination during B and T cell development. V(D)J recombination is tightly regulated in each lineage by cell-type specific mechanisms that drive expression of the RAG1/RAG2 (RAG) endonuclease that catalyzes recombination and activate target AgR loci through the induction of transcription, chromatin accessibility, and chromosome compaction at distinct developmental stages. Moreover, AgR gene assembly is regulated in an allele specific-manner to ensure mono-allelic expression (allelic exclusion) of AgRs via sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. However, a question remains for what prevents recombination of the second allele as the first allele is assembled, expressed, and the protein signals to suppress V recombination. Here, I demonstrate that in primary mouse pre-B cells, RAG DNA double strand breaks (DSBs) introduced on one Igk allele signal via the ATM DNA damage response-kinase and the NFκB essential modulator protein (Nemo) to transiently suppress Rag1/Rag2 transcription, Jk accessibility, and RAG cleavage of the other allele, thereby contributing to Igκ allelic exclusion. Furthermore, I explore RAG DSB responses in primary mouse pro-B and DN cells and identify similar and cell type-specific signals that decrease Rag1/Rag2 transcripts and alter the chromatin activity and structure of Igh and Tcrb loci, respectively. Notably, I observe additional RAG DSB-induced signals targeting the Igh locus in pro-B cells that are consistent with suppression of Igh recombination and may contribute to the more strict enforcement of IgH allelic exclusion as compared to TCRβ. Collectively, my work supports the model of ATM-mediated, RAG DSB-signaled transient feedback inhibition of V(D)J recombination regulating Tcrb and Igh allelic exclusion, and provides a conceptual platform for experiments to elucidate precise mechanisms of mono-allelic AgR gene assembly and the physiological importance of these mechanisms in the pathogenesis of autoimmune disorders and lymphoma.