Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining

Abstract

Summary Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4

“…Using cell lines selectively compromised in different components of NHEJ repair, we have identified a crucial role for LIG4 in mediating long-range inter-chromosomal fusions between damaged telomeres and diverse genomic locations. Consistent with the hallmarks of C-NHEJ (Ghezraoui et al 2014;Oh et al 2014), these telomere-genomic interactions showed reduced microhomology usage at junctions in comparison with intra-chromosomal telomere fusion events. Although the frequency of telomere-genomic events was severely reduced in cells lacking LIG4, there was no analogous impact on intra-chromosomal fusion events, resulting in a notable homogeneity of parameters examined among all samples.…”

“…This result distinguishes intra-chromosomal from inter-chromosomal fusions that demonstrate greater dependency on LIG4 (Ghezraoui et al 2014). …”

DNA double strand break repair pathway choice: a chromatin based decision?

“…Microhomology Mediated End Joining (an alternative NHEJ pathway) leads to the deletion of the sequence between microhomologies (reviewed in 2 ). In addition, recent studies suggest that NHEJ is the primary cause of translocations 3,4 and dysfunctional telomeres fusion. 5 Finally, while HR pathways can be entirely conservative when the sister chromatid is used as a template, dramatic events such as repeat amplification/deletion or loss of heterozygosity (LOH) can occur when HR operates on repeated sequences or homologous chromosomes, respectively.…”

CRISPR-Cas9–guided oncogenic chromosomal translocations with conditional fusion protein expression in human mesenchymal cells

“…DNA double-strand breaks (DSBs) occurring on two chromosomes induce translocations (8)(9)(10)(11)(12), a process primarily mediated by nonhomologous end joining (NHEJ), a nonconservative DNA repair pathway that involves the joining of DNA ends without the involvement of extensive homology (1,13). DSB generation in early model studies was achieved through the introduction of I-SceI endonuclease cleavage sites at two genomic loci, expression of I-SceI, and then selection for the translocation using preintroduced marker fragments (8)(9)(10).…”

“…More recently, the discovery of RNAguided nucleases in bacterial adaptive immunity (CRISPR-Cas9) has facilitated DSB introduction at desired genomic loci (18,19). Using these various programmable nucleases, a number of cancer translocations have been generated in a variety of human cell lines, including EWSR1-FLI1 (ZFNs, CRISPR-Cas9) (11,20,21), NPM1-ALK (TALENs, CRISPR-Cas9) (11,12), and CD74-ROS1 (CRISPR-Cas9) (22). Oncogenic chromosomal inversions have also been engineered in mouse tissues, leading to tumorigenesis (EML4-ALK) (23).…”