184 | Chapter 7 DNA breaks signal to shut down RAG expression to prevent unnecessary DNA damage. That RAG-derived DSBs have an important regulatory function is also illustrated in the recent study of Johnston et al, demonstrating that non-RAG induced DSBs at Ig loci of pre-B cells result in different gene expression profiles than the DSBs instigated by RAG: while other endonucleases, such as Cas9 endonuclease, was also capable of introducing DNA breaks at Ig loci, and triggering the “canonical DDR”, leading to the DNA break repair, measured by the degree of tumor protein p53-binding protein 1(TP53BP1) foci generation, only RAG1/2-mediated DSBs triggered the “non-canonical DDR”, such as processes regulating B-cell development, as measured by the degree of NF-kB2 activation and the CD40 and proviral integration site for Moloney murine leukemia virus 2 (Pim2) expression121. The studies of the RAG regulatory pathways in this thesis underscore how temporal and qualitative circumstances determine the outcome of a signaling pathway modulation: Chapter 5 shows that in cycling pre-B cells AKT signaling regulates RAG expression, while Chapter 4 demonstrates that once the pre-B cells exit the cell cycle, AKT signaling is not involved in the regulation of RAG expression under the DNA damage conditions. Moreover, the mechanisms uncovered in Chapters 5 and 6, where we demonstrated that the regulation of RAG expression involves a negative feedback loop that hinges on the kinase activity of the DNA damage sensor ATM. The degree of crosstalk between these are 2 pathways, (ATM/FOXO1 and ATM/p53/miR-34a/FOXP1), if any, is yet to be determined. We hypothesize that on the one hand, ATM is able to regulate the RAG transcription directly, by binding to Erag and thereby evicting the RAG transcription factor FOXO1 from its binding to Erag, and on the other hand, that ATM activation triggers several regulatory pathways (ATM/FOXO1 or ATM/p53/miR-34a/FOXP1) affecting the availability of the RAG transcription factors. These two effects of ATM activation may co-exist, potentiate each other or fine-tune the expression and activity of RAG1/2 in response to DNA damage.
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