70 | Chapter 3 Discussion In the past, the prevailing thought was that RAG1/2 binding and activity were restricted to regions containing RSSs, such as the immunoglobulin loci, and that this restriction limits the potential genotoxic threat of RAGs. However, in the last decade, it has been shown that RAG1 and RAG2 can bind DNA throughout the genome and that their DNA cleaving activity might not only be limited to the canonical RSS sequences. Using NBS1 as a surrogate marker for DSBs, we identified RAG1/2-specific DSBs in a mouse v-Abl cell line at the genome-wide scale. The off-target activity was examined through differential binding of NBS1 to DNA in RAG-proficient and RAG2-deficient cell lines, which lack the capacity to resolve the DSBs due to the absence of Artemis. This model allowed us to interrogate the DSBs in the chromosomal context of living pre-B cells undergoing Ig recombination. In the previous studies, RAG1 was shown to bind to ~3400 sites in mouse pre-B cells13 and RAG2 to ~18300 sites throughout the mouse genome53. In our study we identified only 1489 putative RAG1/2 DSBs, suggesting that binding or co-localization of RAG1 and RAG2 does not necessarily result in the creation of DSBs. Next to Igk, an accumulation of RAG-specific NBS1 peaks was observed at the Igh locus on chromosome 12. The v-Abl cell line has previously been shown to be arrested in a stage where the Igh recombination has already taken place18. B-cells ensure the preservation of successfully recombined Igh by allelic exclusion, not allowing any subsequent heavy chain recombination on the other allele54. The abundant presence of NBS1 at the Igh region in this experimental set-up suggests that RAG1/2 endonuclease continues to introduce DSBs in this region following the treatment of the v-Abl cells with STI571 and their subsequent transition from pro to pre-B cell stage. Some of the earlier work suggests that v-Abl transformants are able to initiate but not terminate recombination55. It has been suggested that v-Abl antagonizes the pre-B cell signaling pathway and the pre-B cells containing one functional Igh allele continue to transcribe and rearrange the allelic, unrecombined Igh locus, thus not adhering to the allelic exclusion rule56. In addition, subsequent work showed that B cells with functional B-cell receptors may continue the recombination and “edit” their specificity, allowing the cells with auto-reactive receptors to escape their elimination57. The off-target activity of RAG1/2 has been associated with a number of genomic lesions often seen in B-ALL cases. Though our data showed RAG-dependent NBS1 peaks in the proximity of genes associated with B-cell malignancies, the genes that are most frequently translocated in B-ALL (ETV6/RUNX1 or BCR/ABL1) or deleted (E2A, EBF1, LEF1, IKZF1 or IKZF3)5–7,11 were not identified in our model. This could be attributed to several factors, for instance, differences in murine and human chromatin configuration, or to the fact that the v-Abl B cell line has already acquired the transforming genomic event, which may have changed the accessibility of some of the genes that are frequently translocated in human patients. Furthermore, only 2.7% of the RAG1/2-dependent NBS1 peaks (SUPPL
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