2 Role of RAG1 and RAG2 in B-cell development | 37 MDM4, and E2F1186–188. The Chk2-mediated stabilization of p53 drives the transcription of p21CIP and halts the progression of the cell cycle through inhibition of CDKs189. In developing B cells, the RAG1/2-generated DSBs were shown to activate apoptotic factors that promote cell survival to enable additional V(D)J recombination events, for instance, Igk-derived DSBs were shown to promote p53-mediated activation of the pro-apoptotic BAX protein as well as the ATM-mediated up-regulation of the pro-survival protein Pim2190. The balance between the pro-apoptotic and the pro-survival signals determines the outcome. Our lab demonstrated that in response to external or RAG-derived DSBs, several related negative feedback mechanisms are triggered that limit the availability of RAG1 and RAG2, thereby dampening the V(D)J recombination activity, and thus preventing further genomic instability. The activation of ATM, as a response to external, but also to the RAG-created DNA breaks, resulted in the rapid downregulation of RAG1 and RAG2 expression. This downregulation was shown to be mediated on one hand through the ATM-dependent release of FOXO1 from binding to Erag, and on the other hand by ATM-dependent reduction of FOXO1 levels. Strikingly, PI3K/AKT did not show any involvement in the DNA damage-mediated downregulation of RAG1/2, suggesting under the genotoxic stress conditions, this pathway does not regulate RAG expression, unlike observed under the normal, non-stressed conditions121. In addition, our work also indicates that the ATM-mediated activation of p53 constitutes an additional regulatory circuit, resulting in decrease of FOXP1 levels, possibly through the p53-dependent activation of microRNA-34a (miR-34a), which targets and downregulates FOXP1 expression, and thereby limits the expression of RAGs in response to external and RAG-mediated DSBs (Ochodnicka-Mackovicova et al, manuscript under consideration). In this negative feedback regulation, the RAG-dependent DSBs limit themselves and protect the genomic stability of B cells from excessive RAG activity (Figure 4). The RAG-induced DSBs introduced on one Igk allele suppress, through ATM signaling, RAG expression and thereby suppresses RAG-mediated cleavage on the other allele, contributing to Igk allelic exclusion. It was demonstrated that immature B cells from ATM-deficient mice demonstrated increased frequencies of bi-allelic cleavage at Igh and Igl. It was proposed that the activation of ATM results in the down-regulation of growth arrest and DNA damage-inducible alpha (GADD45a), which subsequently represses its targets Rag1 and Rag236,191. It has been proposed that in this way, the RAG activity creates a negative feedback loop limiting its own expression and protecting the genomic stability, though in our previous study, the genotoxic stress in mouse v-Abl-transformed pre-B cells did not confirm the ATM-dependent effect on the levels of GADD45a121. In eukaryotic cells the DSB-prompted ATM activation initiates phosphorylation of the histone variant H2AX at Ser139, giving rise to so-called gH2AX. gH2AX accumulates in the proximity of the DSBs and serves as a landing pad for other components of the DDR cascade192. In murine cells, gH2AX promotes the hairpin processing by Artemis. In the absence
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