12 | Chapter 1 sistent RAG1/2 activity, thereby potentially increasing the occurrence of genomic lesions resulting in oncogenic events. In chapters 4, 5, and 6 we describe the interplay between DNA breaks and RAG1/2 activity and regulation. In Chapter 4 we showed that extrinsic DNA damage leads to the downregulation of RAG1/2 expression in pre-B cells. We identified the ataxia telangiectasia mutated (ATM) kinase as a key player in the DNA damage-dependent regulation of RAG1/2 transcription, which primarily impinges on the transcription factor forkhead box O1 (FOXO1). In Chapter 5 we showed that the nuclear factor kappa-B (NF-kB) and phosphoinositol-3 kinase (PI3K)/ AKT signaling pathways act in concert to suppress inappropriate RAG1/2 expression and activity in human and mouse pre-B cells and in B-cell acute lymphoblastic leukemia (BALL) patients. Finally, in Chapter 6 we identified an additional regulatory mechanism that regulates RAG1/2 expression in response to DNA damage, involving the p53-dependent expression of microRNA-34a (miR-34a), which acts by inhibiting forkhead box protein P1 (FOXP1), a transcription factor that drives RAG1/2 expression. In summary, the studies presented in this thesis show that RAG1/2 is capable of introducing DNA breaks outside of the Ig loci, located in the proximity of SSR sequences. In addition, our studies show that DNA breaks in developing pre-B cells also have an important regulatory function by instigating regulatory feedback loops that limit the expression and activity of RAG1/2, thereby contributing to safeguarding genome stability and integrity in developing B cells. These findings are further summarized and discussed in Chapter 7, outlining their potential implications and perspectives.
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