7 General discussion | 173 growth response 1 (EGR1), or Ras responsive element binding protein 1 (RREB1)28–30, as well as modulators of p53 signaling and apoptosis, such as POZ/BTB And AT Hook Containing Zinc Finger 1 (PATZ1) or specificity protein 1 (SP1)31,32. PRDM9 is a zinc finger protein with histone methyltransferase activity, catalyzing the methylation of lysine (K) 4 of the H3 histone (H3K4me3), primarily during meiosis33. Interestingly, the H3K4me3 histone epigenetic mark was associated with open chromatin configuration and with transcriptionally active promoters of genes regulating cell growth, cell migration, or angiogenesis, all of which makes H3K4me3 an important regulator in cancer immunity34,35. EGR1 is a zinc-finger transcription factor regulating cell growth, differentiation and participating in the regulation of B-cell development36. Another identified motif was linked to the MYC-associated zinc-finger protein (MAZ), a transcription factor known to regulate the expression of MYC oncogene and affect the large-scale genome organization through its interaction with cohesins37,38. The off-target RAG activity in the aforementioned regions poses a potential threat of introducing a genomic lesion in these genes, making pre-B cells susceptible to malignant transformation. Another recurrent motif type observed in our study was simple dinucleotide sequence repeats, such as CA and GA or TA repeats. Our supervised motif analysis of the CA and GA repeats revealed strong enrichment in the regions 500-1000bp around (5’ and 3’) RAG-dependent NBS1 peaks. Naturally occurring repetitive sequences have been demonstrated to assume non-canonical DNA structures, so-called non-B DNA. These structures include G-quadruplexes, which are guanine-rich sequences, H-DNA which are mirrored repeats of oligopurine sequences forming triplex DNA, Z-DNA which are AT/GC-rich sequences, or cruciform and other hairpin-forming DNA structures. Such structures are known for adopting an open chromatin configuration and for affecting DNA replication and transcription, which makes them more susceptible to genomic instability39,40. In the context of hematological malignancies, non-B DNA structures were observed in the proximity of the breakpoints of the hallmark genomic lesions. For instance, in Burkitt’s lymphoma and in mouse t(12;15) BALB/c plasmacytomas, H-DNA forming regions have been identified near the breakpoints of the translocated c-myc locus. The presence of non-B DNA structures has also been demonstrated in the proximity of the major breakpoint region (Mbr) of Bcl-2 in follicular lymphoma41. Notably, RAG1/2 has exhibited the ability to cleave non-B DNA structures in the absence of RSS, acting as a structure-specific endonuclease. In fact, CA repeats were shown to mimic cRSS with their nonamer and heptamer elements, and have been identified as specific targets of RAG1/2 activity. Consequently, RAG1/2 inadvertently introduced DNA breaks at CA-rich sites42,43. In addition, a more recent study highlighted the essential role of the zinc-binding domain (ZnC2) of RAG1 in its binding to non-B DNA structures. Mutations in the ZnC2 domain have been found to impede the RAG’s nicking capacity on DNA G-quadruplexes and on the single-stranded DNA regions44.
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