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Researchers identify points where incorrect immune cell DNA editing causes blood cancer.

Antigen receptor genes are assembled by controlled, large-scale deletions and inversions. Errors in this process, known as V(D)J recombination, produce aberrant genomic rearrangements, which can fuel the development of T- and B-cell malignancies.

Now, a study from researchers at the University of Pennsylvania have shown that when V(D)J recombinase, hits so-called ‘off-target’ spots on a chromosome, the development of immune cells can lead to cancer in animal models. The team state that knowing the exact nature of these editing errors will be helpful in designing therapeutic enzymes based on these molecular scissors. The opensource study is published in the journal Cell Reports.

Earlier studies from the team showed that V(D)J recombinase (consisting of the RAG1 and RAG2 proteins) normally sends a break in DNA down the correct repair path by preventing access to other, inappropriate repair mechanisms. This shepherding process can be disabled if the ‘C’ terminus of the RAG2 protein subunit is removed. This causes genomic instability in developing immune cells and, in the absence of a working tumour suppressor protein such as p53, an aggressive form of lymphoma developed in mice.

In the current study, genome-wide analysis of lymphomas of the thymus in these mice with the truncated Rag2 protein revealed a surprise, that numerous off-target DNA rearrangements caused these deletions.  Previous findings from the lab had suggested that a different mistake, chromosome translocations, or swaps between two chromosomes, might underlie the development of lymphoma in these mice, however whole genome sequencing revealed deletions as the main drivers of these cancers.  Results show that these rearrangements affected several known and suspected oncogenes and tumour suppressor genes, including Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9.

Data findings show that genome-wide analysis of chromatin marks also suggested that normal interactions between the C-terminus of the Rag2 protein subunit and a specific chromatin modification helps maintain the fidelity of DNA target recognition by the enzyme. Results show that gene expression is regulated by epigenetic methylation and acetylation. The team explain that certain chemical groups on histones allow DNA to open up, and others to tighten the chromatin, and so creating availability to be read and expressed as proteins.

The researchers surmise that the cancer-causing effects of off-target deletions mistakenly created by the V(D)J enzyme needs to be considered in designing site-specific enzymes for genome modification such as zinc-finger nucleases, TALENS, or CRISPRs.  For the future, the lab conclude that it might not be a simple task to computationally predict the unintended targets of manmade genome, the strategies used in the V(D)J recombination system may simplify and speed these efforts.

Source: Perelman School of Medicine, University of Pennsylvania

Genome-wide analysis of thymic lymphomas from Tp53−/− mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification. Off-Target V(D)J Recombination Drives Lymphomagenesis and Is Escalated by Loss of the Rag2 C Terminus. Lewis et al 2015.
Genome-wide analysis of thymic lymphomas from Tp53−/− mice with wild-type or C-terminally truncated Rag2 revealed numerous off-target, RAG-mediated DNA rearrangements. A significantly higher fraction of these errors mutated known and suspected oncogenes/tumor suppressor genes than did sporadic rearrangements (p < 0.0001). This tractable mouse model recapitulates recent findings in human pre-B ALL and allows comparison of wild-type and mutant RAG2. Recurrent, RAG-mediated deletions affected Notch1, Pten, Ikzf1, Jak1, Phlda1, Trat1, and Agpat9. Rag2 truncation substantially increased the frequency of off-target V(D)J recombination. The data suggest that interactions between Rag2 and a specific chromatin modification, H3K4me3, support V(D)J recombination fidelity. Oncogenic effects of off-target rearrangements created by this highly regulated recombinase may need to be considered in design of site-specific nucleases engineered for genome modification. Off-Target V(D)J Recombination Drives Lymphomagenesis and Is Escalated by Loss of the Rag2 C Terminus. Lewis et al 2015.

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