7 General discussion | 183 light chain recombination, similar to the increase observed in Tp53-/- primary pre-B cells, suggesting that more pre-B cells have undergone Igk recombination. In addition, the absence of RAG1, and thus absence of RAG-dependent DSBs in primary pre-B cells coincided with higher Foxp1 mRNA levels. We propose that the increase in Foxp1 levels might be related to the lack of gene recombination-mediated activation of p53 DNA damage responses, including the miR-34a. In search of a more detailed mechanistic explanation of the DNA damage-driven RAG1/2 regulation, we speculate about several possible scenarios: The pro-survival factor B-cell leukemia/lymphoma 2 (BCL2) has also been identified as one of the transcripts downregulated upon overexpression of miR-34a. Interestingly, BCL2 has been shown to partially compensate for the loss of Vpr-binding protein (VprBP, also known as DCAF1), which is a substrate adaptor protein, associating with the N-terminus of RAG1 and is required for B-cell development and V(D)J recombination119,120. Whether in our model the effect of the loss of p53 or miR-34a on the amount of B220+DX6-Ly6C-IgM+ B cells following the IL7 withdrawal could be (partially) attributed to BCL2 remains an open question. Another possible mechanistic explanation could be provided by the study showing that in the induction of miR-34a expression by cisplatin treatment downregulates Sirtuin 1 (Sirt1), which in turn increased the NF-kB activity116. Previously, we have established that in cycling pre-B cells, the NF-kB pathway inhibits RAG1/2 expression through AKT and CDK483. Whether the DNA damage-driven expression of miR-34a in pre-B cells could contribute to RAG1/2 downregulation through NF-kB activation is yet to be established. In summary, we propose a negative feedback regulatory mechanism involving p53, miR-34a, and FOXP1, whereby DNA damage limits the expression of RAG1 and RAG2. In this regulatory pathway, DNA damage (exogenous as well as RAG-induced) triggers the activation of ATM, which subsequently stabilizes p53 and initiates the downstream effects of p53, including the upregulation of miR-34a. MiR-34a then modulates the levels of FOXP1 and the recombination of Igk in pre-B cells. Consistent with this model, both miR-34a and FOXP1 levels respond to both exogenous and RAG-induced DNA damage, thereby constraining the expression of RAG1/2 and V(D)J recombination. This regulatory mechanism in developing B cells likely functions as a self-protective mechanism against excessive RAG-induced DNA damage, thereby safeguarding genome stability. Final thoughts The RAG-mediated DSBs are vital for the assembly of BCR (and TCR), without which our immune system would not be able to adapt and to protect us from the armada of different pathogens, they are also seen as a potentially hazardous product of RAG1/2 activity. Research data in this thesis indicate that the RAG-induced DSBs also display regulatory capacities and are capable of triggering a self-regulatory mechanism, where the RAG-produced
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