4 DNA damage response regulates RAG1/2 expression through ATM-FOXO1 | 99 1 (NBS1)44,45 was readily detected (Figure 5D). The fact that we could ChIP phospho-ATM at the Erag enhancer region in NCS-treated cells whereas FOXO1 DNA-binding was abolished under these conditions (Figure 2D) suggests that ATM may locally regulate/impair the DNA occupancy of FOXO1, but most likely this does not involve direct phosphorylation of FOXO1 by ATM. Of note, the interaction between phosphorylated ATM and FOXO1 was already observable in STI571-treated cells and was further increased after induction of DNA damage. This suggests that there is already some degree of DNA damage in cells treated with STI571. Since RAG expression is induced by STI571 we proposed that RAG-mediated DNA cleavage could be the source of this DNA damage, thereby activating negative feedback regulation of RAG expression. RAG-dependent DNA breaks regulate RAG1 expression through a negative feedback mechanism To assess whether the RAG-dependent endogenous DNA breaks are able to regulate RAG1 expression, WT (A70) and Rag2-/- (R2K3) mouse v-Abl cells were treated with STI571 for 72 hours and Rag1 mRNA expression was quantified. Because both RAG1 and RAG2 are needed to form the RAG1/2 complex that possesses the endonuclease activity, no Ig rearrangement-related DNA breaks are generated upon STI571-treatment in Rag2-/- v-Abl cells27. Interestingly, Rag1 mRNA expression was 2.7-fold higher in STI571-treated Rag2-/- as compared to WT v-Abl cells (Figure 6A). Inhibition of ATM kinase activity increased Rag1 mRNA expression in WT v-Abl cells to a similar level as observed in Rag2-/- v-Abl cells, but did not alter Rag1 mRNA expression levels in Rag2-/- v-Abl cells. In accordance, Rag2-/- v-Abl cells showed enhanced Erag-Rag1 luciferase reporter activity compared to WT v-Abl cells upon STI571 treatment (Figure 6B). To further substantiate the point that DNA breaks can regulate the Rag1 expression levels in Rag2-/- v-Abl cells, we show that exogenously induced DNA breaks by NCS also provoked the ATM-dependent inhibition of Rag1 transcription in these cells (Figure 6C). In addition, we show that in the absence of Artemis nuclease, which is required for the repair of RAG1/2-dependent coding joint hairpins, Rag2-deficient cells have significantly increased Rag1 mRNA expression compared to Rag2-proficient cells (Figure 6D). Also, the expression level of Rag1 mRNA is about twofold lower in STI571-treated Artemis-/- versus wildtype v-Abl cells (compare Figures 6A and 6D), which may be caused by the accumulation of RAG1/2-dependent breaks in Artemis-deficient cells, due to the lack of repair of coding ends.
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