Project Summary: B cell-mediated adaptive immunity relies on the programmed induction of DNA double strand breaks (DSBs) to create diverse immunoglobin (Ig) gene repertoires. The RAG1/RAG2 (RAG) endonuclease assembles Ig genes through recombination of variable (V), diversity (D), and joining (J) gene segments of Ig loci. This process is vital for adaptive immunity and mammalian survival yet, it also confers risk, as most B lineage cancers contain clonal translocations involving an Ig locus and a proto-oncogene, underscoring the vital importance of tightly regulating V(D)J recombination. My thesis lab discovered that RAG or genotoxic DSBs rapidly repress transcription of the Rag1/2 locus by signaling via the ATM kinase, a key regulator of the cellular DSB response. Atm-/- mice have higher frequencies of developing B cells with RAG DSBs at both Ig alleles and of mature B lymphocytes with Ig translocations. Moreover, B cell-specific Atm deletion in mice increases the incidence of B lineage lymphomas with Ig translocations. These data are consistent with DSB-induced repression of Rag1/2 being critical to protect from Ig translocations; yet, given the multifunctional roles of ATM in the DSB response, these phenotypes cannot be directly attributed to DSB-induced repression of Rag1/2 expression. Genotoxic DSBs induce ATM-dependent phosphorylation of the NFκB essential modulator (Nemo) protein to activate NFκB transcription factors. I show that Nemo-/- developing B cells have impaired repression of Rag1/2 in response to DSBs and display increased RAG DSBs at Ig loci. My data are consistent with prior Atm-/- studies, but unlike Atm-/- cells, Nemo-/- cells retain normal DSB repair and checkpoint/apoptosis activation. Thus, the Nemo-/- model provides me an opportunity to more directly test my central hypothesis that DSBs induce Nemo/NFκB-mediated transcriptional repression of Rag1/2 to suppress Ig translocations and resultant lymphoid cancers. I propose to elucidate the mechanism of Nemo-dependent Rag1/2 repression. Aim 1 of this proposal will test the hypothesis that NFκB factors directly bind the Rag1/2 Erag enhancer to mediate DSB-induced repression of Rag1/2 by inhibiting transcriptional elongation. This may reveal novel mechanisms of NFκB- mediated transcriptional repression, which are largely undefined. I will determine DSB-induced NFκB binding sites and their functional role in Rag1/2 repression. Furthermore, I will define the transcriptional state of Rag1/2 in the absence or presence of DSBs to determine the mechanistic levels at which DSBs repress Rag1/2. Aim 2 will test the hypothesis that DSB-induced repression of Rag1/2 limits RAG DSBs and thereby suppresses oncogenic Ig translocations. Mice with B cell specific deletion of Nemo will be used to i) quantify RAG DSBs at Ig alleles in pro-B and pre-B cells, ii) quantify Ig translocations in non-malignant B lineage cells, and iii) evaluate predisposition to B lineage cancers with oncogenic Ig transl...