56 | Chapter 3 accumulation of RAG1 and RAG2 was demonstrated near the cryptic RSS (cRSS), which are sequences that structurally resemble the canonical RSS. Interestingly, no endogenous recombination events were detected in the tested gene pairs of thymocyte genomes, concluding that despite the promiscuous RAG1 and RAG2 binding, recombination events must be rather rare13,14. In extrachromosomal V(D)J recombination assays, or cell systems with artificially introduced cRSS-containing constructs, RAG1/2 was demonstrated to bind and cleave the non-Ig loci13,15,16. However, the off-target activity of RAG1/2 in endogenous chromosomal V(D)J recombination context in pre-B cells is less well described. In this study, we aimed to identify off-target RAG1/2-dependent DSBs on a genome-wide scale in in vivo endogenous chromosomal V(D)J recombination context, using pre-B cells that can be induced to undergo immunoglobulin kappa light chain (Igk) recombination, expressing endogenous levels of RAG1/2, but that are unable to repair the DSBs. We made use of Artemis-deficient mouse v-Abl/Em-Bcl2 pre-B cells where Artemis deficiency prevents the hairpins of the coding ends from being opened, thus leading to accumulation of the RAG-dependent DNA breaks that would otherwise be quickly resolved by c-NHEJ. We used the DNA-bound repair protein NBS1 as a proxy of coding ends. NBS1, being a member of the MRN DNA repair complex, binds the DNA in close proximity to DSBs. Performing an NBS1 chromatin immunoprecipitation followed by massive parallel sequencing (ChIPSeq) in the pre-B cells undergoing Igk recombination allows genome-wide determination of the position of DSBs, as has previously been demonstrated in a study investigating the off-target AID-derived DNA breaks in splenic B cells17. Comparing the NBS1 ChIP-Seq profiles of RAG-proficient pre-B cells to the RAG2-deficient cells allowed us to select and analyze the RAG-dependent DSBs. Using this approach, we identified 1489 RAG1/2-dependent DSBs throughout the genome. One of the common features of the off-targets is the presence of simple sequence repeats in the proximity of the putative RAG off-targets, further underscoring the vulnerability of such regions to DNA damage. Material and methods Cell culture and small molecule compounds Mouse v-Abl transformed B-cell lines were used as a model to study the off-target activity of endogenous RAG1 and RAG2. The v-Abl-transformed cells are characterized by the surface expression of B220+CD43+CD25−IgM−, which is typical for B cells in the pro- or early pre-B cells stage. At this stage, the B cells proliferate rapidly and express very little or no RAG1 and RAG2. Also, their Igk at this stage had not been yet rearranged. Treatment with Abelson kinase inhibitor (STI571/Imatinib), leads to G1 arrest, upregulation of RAG1 and RAG2, and initiation of Igk recombination18. The treatment with STI571 also triggers an apoptosis within several hours. As such cell system would be sub-optimal to study the (off)-targeting activity of RAG1/2, the cell lines also carry an Eμ-Bcl2 transgene, allowing
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