28 | Chapter 2 no further modifications of the joint90. An in vitro cell-free RAG activity assay showed that the truncated but catalytically active forms of RAG easily catalyzed the formation of hybrid joints and open-shut joints suggesting that the full-length forms repress their formation. The in vitro transposition activity of full-length RAG2 was dramatically reduced as opposed to the full-length RAG1 transposition activity91. Several studies have shown that the ESCs do not seem to be as inert as initially thought, and in fact, in various in vitro assays they display the capacity to re-integrate elsewhere in the genome. However, it is supposed that the in vivo threat to the integrity of the genome caused by RAG-mediated transposition is rather limited. Only a few in vivo RAG-mediated transposition events have been described to date, and the evidence linking leukemia or lymphoma cases to RAG-mediated transposition events remains inconclusive86,92–95. The RAG/ESC complex could, so far only theoretically, catalyse the formation of DSBs as it still contains two RSSs (Figure 2A). It has been observed that upon cleavage of the ESC is opened at the RSS but the RAG1/2, which is still bound to the ESC, is able to introduce DSBs at the next RSS, a process termed as “cut-and-run”. Recently, a next-generation genome-wide sequencing of the leukemic B cells revealed similarities between the “cut-andrun” breakpoints and the breakpoints observed in B-cell leukemia that harbor the ETV6/ RUNX1 chromosomal translocation96,97. These studies argue that the lack of evidence is caused by the limitations of the previously used techniques and suggest that the advances in the next-generation sequencing techniques of the whole genome could provide a much better resolution of the genomic events, and thus in the future deliver the missing evidence of RAG-mediated ESCs re-integration in lymphoid malignancies98,99. RAG mistargeting Sequences that resemble RSS, termed cryptic RSS (cRSS), were shown to be very abundant in the vertebrate genome, with a remarkable estimate of one cRSS per each 600bp100. A study employing genome-wide chromatin immunoprecipitation (ChIP) of RAG1 and RAG in mouse pre-B cells, followed by next-generation sequencing (ChIP-seq), clearly demonstrated that RAG1 and RAG2 binding in the genome is not solely limited to the Ig loci, as around 3400 RAG1 and around 18300 RAG2 binding sites were identified throughout the genome of mouse pre-B cells. However, the presence of RSS alone seems to be a poor indicator of RAG1-binding sites; the heptamer and cRSS sequences were even found to be depleted from the RAG1-binding sites. Also, in this study no translocations were detected of the selected examples of genes with high content of cRSS in their proximity, concluding that RAG1/2 binding outside of the Ig/Tcr loci only rarely results in translocation events71. On the other hand, the RAG2 binding throughout the lymphocyte genome coincided significantly with regions containing high levels of histone H3 trimethylated at lysine 4 (H3K4me3)101,102, which was not entirely surprising considering that the PHD finger of RAG2 was shown to specifically recognize and bind H3K4me3 in mammalian cells. Mutations
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