7 General discussion | 175 next G1 phase. RAG2 regulation at the G1-S transition has been attributed to cyclin-A/ cyclin-dependent kinase 2 (CDK2) which phosphorylates RAG2 at Thr490. This facilitates the formation of an S-phase kinase-associated protein 2 (SKP2) binding site, which ultimately results in ubiquitylation and degradation of RAG249,50. While CDK2 was shown to trigger proteasomal degradation of RAG2, RAG1 stability appears independent of the cell cycle51–53. Interestingly, we observed that DNA damage negatively affects the protein stability of RAG1, as in human v-Abl transformed pre-B cells co-treatment with NCS and the protein synthesis inhibitor cycloheximide (CHX) shortened the RAG1 half-life as compared to CHX-only treatment. The N-terminus of RAG1 contains a functional E3 ubiquitin ligase domain called Really Interesting New Gene (RING)54, which is known to catalyze the ubiquitination of H3, but also of RAG1 itself55,56. Recently, it was discovered that mouse mutants carrying P326G mutation in the RAG1 RING domain accumulate RAG1 protein, probably due to the inability to degrade RAG by autoubiquitylation57. We further demonstrated that the DNA damage-induced RAG1 protein downregulation cannot be attributed to damage-induced caspase cleavage, leaving the mechanism of the DNA damage-induced shortened RAG1 protein half-life incompletely understood with an open possibility for ubiquitin-mediated regulation. Recently, the Swanson group reported that VprBP/DCAF1, functioning as a substrate adaptor within the cullin-RING E3 ubiquitin ligase 4 (CRL4) ubiquitin ligase complex, plays a crucial role in regulating the levels of RAG1 protein in murine B cells, facilitating the turnover of RAG1. In vivo depletion of VprBP/DCAF1 leads to increased expression of RAG1, resulting in excessive V(D)J recombination and a skewed repertoire of Ig light chains58,59. It is therefore conceivable that a part of the DNA damage-induced rapid loss of RAG1 protein is conveyed by a reduction of RAG1 protein stability. However, whether VprBP/DCAF1 responds to DNA damage and whether it could be the missing link for a mechanistic explanation of DNA damage-induced reduction in RAG1 protein stability is yet to be investigated. The regulation of Rag1 and Rag2 gene transcription involves various transcription factors binding to cis-acting sequences, mainly FOXO1 and FOXP1 binding the Erag enhancer, inducing RAG1 and RAG2 expression during B-cell development60,61. We therefore investigated whether the DNA damage-induced RAG1/2 downregulation is linked to transcriptional regulation by FOXO1. Our study shows that DNA damage abolished FOXO1 binding to Erag enhancer, and that the ATM kinase inhibition is able to restore the FOXO1 binding to Erag, linking both ATM and FOXO1 to the DNA damage-induced downregulation of RAG1. Moreover, we found that DNA damage-activated ATM phosphorylated at Ser1981 was bound to Erag, possibly locally releasing FOXO1 from binding to Erag. Specifically, the phosphorylation of FOXO1 on Ser215 has previously been shown to be required for FOXO1 binding to Erag, and the FOXO1 Ser215 phosphorylation was demonstrated to be dependent on MAPK-activated protein kinase (MAPKAPK5)62. We could not investigate the effect of DNA damage on FOXO1 Ser215, nor its binding to Erag, due to the unavailability of reagents
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