62 | Chapter 3 Figure 2. (A) NBS1 ChiP-Seq in Artemis-/- cells (AH2) and Artemis-/-Rag2-/- (R2K.Artm) cells showing Igk locus at chromosome 6. Distinct accumulation of reads in the AH2 cell line, and their absence in R2K.Artm cell line in this region, suggests that NBS1 binding, and thus the presence of DSBs at this locus is only found in the Rag-proficient cell line. Input ChIP-Seq sample shows the background level of the mapped reads. (B) and (C) examples of 2 NBS1 peaks outside of the Ig loci: chromosome 4 (147160850-147160999) and chromosome 14 (39423000-39423149), where AH2 shows the NBS1 reads in RAG-proficient cell line, R2K.Art shows the NBS1 reads in the RAG-deficient cell line and an untreated input sample shows the background levels of NBS1 in these regions. In STI571-treated v-Abl cells, the Igk locus undergoes gene rearrangement, which was reflected in the frequency of RAG-dependent NBS1 peaks in this region, which was 8.8fold higher (4.85 breaks per million bp) than the frequency of the RAG-dependent NBS1 peaks throughout the whole mouse genome (0.55 breaks per million bp; p<0.001). Interestingly, we also found a hotspot at the Igh locus on chromosome 12, showing 33.1 fold higher frequency of RAG-dependent NBS1 peaks (18.2 peaks per million bp; p<0.001), as compared to 0.5 peaks per million bp in the rest of the genome (SUPPL Table 1A), which is a 33-fold difference (p<0.001), suggesting simultaneous RAG1/2 activity at Igh and the immunoglobulin light chain (Igl). This is rather unexpected as the v-Abl transformed pro-B cells have previously been shown to contain rearranged Igh, which should prevent any subsequent recombination on this locus18. RAG1/2 induces DNA breaks outside of Igk locus Using our approach, 1489 RAG-dependent NBS1 peaks were identified throughout the genome, 95,3% of which were located outside of the Ig loci (Figure 2B and Figure 2C are examples of RAG-specific NBS1 peaks identified outside of the Igk locus, SUPPL Table 2 for a complete overview of the RAG-specific NBS1 peaks). Interestingly, some of the RAG-specific NBS1 ChIP-Seq peaks, next to the 2 hotspots (Igk and Igh), were localized in or near
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