7 General discussion | 169 Key findings of this thesis: 1. The RAG1/2 endonuclease complex is able to introduce DNA breaks outside of the Ig loci in mouse developing B cells. Using a genome-wide approach, RAG-dependent DNA breaks were mapped in the mouse genome. The motif analysis revealed that simple repeat sequences and GC-rich regions were identified in the proximity of the RAG1/2-dependent DNA breaks. The simple repeat sequences and GC-rich regions are known to form alternative DNA structures. We propose that in the genome-wide context, such structures are targeted by the RAG1/2 endonuclease complex. (Chapter 3) 2. DNA breaks trigger a negative feedback loop that limits RAG1/2 expression in developing B cells. In response to external and RAG1/2-derived DNA damage in pre-B cells, the DNA damage sensor ataxia telangiectasia mutated (ATM) negatively regulates the RAG1/2 transcription factor forkhead box protein O1 (FOXO1), resulting in downregulation of RAG1/2. This describes how DNA breaks, which result from the activity of RAG1/2, control the expression of RAG1/2. (Chapter 4). 3. Several layers of regulation ensure genome stability by restricting RAG1/2 activity to the G1 cell cycle phase in developing lymphocytes. In proliferating pre-B cells, RAG1/2 expression and activity are suppressed, preventing collateral DNA damage. We identified a novel regulatory mechanism suppressing RAG1/2 in proliferating pre-B cells. The nuclear factor kappaB protein (NF-kB) and phosphoinositide-3 kinase (PI3K /AKT) signaling pathways suppressed RAG expression in cycling mouse and human pre-B cells. Inhibition of NF-κB resulted in increased RAG expression, and recombination activity, and led to RAG-dependent DNA damage in proliferating pre-B cells. (Chapter 5) 4. DNA breaks trigger the DNA damage response (DDR) with the tumor suppressor p53 being a central downstream molecular mediator. We have identified a novel regulatory pathway in which activation and stabilization of p53 suppressed RAG expression in mouse pre-B cells. This regulatory mechanism involved the p53 target miR-34a and the RAG1/2 transcription factor forkhead box protein P1 (FOXP1). We propose that in this way the DNA breaks limit RAG1/2 expression, thus protecting the pre-B cell genome stability. (Chapter 6) RAG1/2 off-target DNA cleavage in developing B cells During B-cell development, gene recombination follows a specific order of events to ensure the creation of a diverse repertoire of immunoglobulins. Initially, the immunoglobulin heavy chain (Igh) locus undergoes recombination, where V(D)J gene segments
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