6 DNA damage-induced p53 downregulates expression of RAG1 | 149 Results and discussion In developing pre-B cells, V(D)J rearrangement is orchestrated by the RAG1/2 endonuclease complex. Aberrant regulation of this rearrangement process has been implicated in the development of B-cell malignancies, such as B-cell acute lymphoblastic leukemia (B-ALL) and follicular lymphoma (FL)1–3. Persistent expression of RAG1 was shown to be an oncogenic driver in B-ALL4 and RAG1/2 regulators have been suggested as plausible therapeutic targets5–7. The double-stranded DNA breaks (DSBs) created by RAG activate the ATM kinase, which then recruits and activates other proteins to the site of the breaks in the efforts of its repair. Following the Ig kappa light chain (Igk) recombination on one allele, the RAG-dependent DNA breaks activate ATM, which was shown to limit RAG expression and accessibility of the Igk locus, thus enforcing allelic exclusion8,9. Previously, we have demonstrated that in response to external DNA damage, RAG protein and mRNA levels are rapidly downregulated, and that the regulation involves the ATM-FOXO1 pathway, where pharmacological inhibition of ATM prevented the DNA damage-induced downregulation of Rag1 and Rag27. To further validate the involvement of ATM in the DNA damage-dependent regulation of RAG we expanded progenitor B cells from Atm-/- mouse bone marrows by IL7 and Flt3L stimulation in vitro. The large-to-small pre-B cell transition was induced by IL7 and Flt3L withdrawal. In agreement with our previous findings7, we show that IL7/Flt3L withdrawal induced Rag1 and Rag2 expression, which was downregulated upon 5 Grays (Gy) of g-irradiation in wildtype (WT) pre-B cells, but not in Atm-/- pre-B cells (Figure 1A-C). ATM activation is known to stabilize TP53 via a complex downstream array of events, including phosphorylation of mouse double minute 2, also known as E3 protein ubiquitin ligase, (MDM2), which in its phosphorylated form cannot poly-ubiquitinate p53, thus leading to its stabilization10,11. Here, we show that the ATM-mediated regulation of Rag also involves p53. Rag1 and Rag2 mRNA was rapidly downregulated in response to external DNA damage in mouse primary pre-B cells, with on average 89.9% reduction in Rag1 and 90.7% in Rag2 mRNA levels in 5 Gy-irradiated primary WT pre-B cells, whereas in Tp53-/- mouse primary pre-B cells g-irradiation resulted in an on average 44.3% reduction in Rag1 and 25.6% in Rag2 mRNA levels (Figure 1D-E). In addition, in primary bone marrow pre-B-cell cultures prompted to undergo Igl recombination by IL7 and Flt3L withdrawal from the culture, the amount of surface IgM-expressing immature B cells (B220+DX6-Ly6C-IgM+) was higher in Tp53-/- cultures cells as compared to their WT counterparts (1.32% vs. 0.79%, respectively), suggesting that p53 activation interferes with the successful production and expression of surface IgM in developing B cells (Figure 1F).
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