Major finding: In contrast to mice, human cells require canonical NHEJ for DSB-induced chromosomal translocations.
Approach: DNA nucleases were used to induce translocations in human cells lacking NHEJ components.
Impact: Species-specific mechanisms generating chromosomal rearrangements likely influence carcinogenesis.
Reciprocal chromosomal translocations are frequently observed in cancer cells and are responsible for generating oncogenic fusion genes as well as driving proto-oncogene expression. Previous studies have shown that, in murine cells, the canonical nonhomologous end-joining (NHEJ) pathway suppresses chromosomal translocations, which are instead mediated by alternative NHEJ. Interestingly, however, breakpoints characterized in human cells do not always display significant regions of microhomology, which are typically associated with alternative NHEJ activity, prompting Ghezraoui and colleagues to study the relative contribution of canonical and alternative NHEJ in chromosomal translocations in human cells. To address the role of the canonical NHEJ pathway in translocation formation, a series of zinc finger and transcription activator–like effector (TALE) nucleases designed to generate site-specific double-strand breaks (DSB) were expressed in a variety of human cell lines deficient for the canonical NHEJ components DNA ligase IV (LIG4) or X-ray repair cross-complementing protein 4 (XRCC4). Analysis of the resulting junctions showed that loss of canonical NHEJ led to reduced translocation frequency, longer deletions, and larger regions of microhomology. Similar experiments performed using patient-derived fibroblasts that harbor hypomorphic LIG4 mutations revealed that reduced canonical NHEJ activity led to an intermediate translocation phenotype. Expanding on these findings, Cas9-induced DSBs or paired DNA nicks, but not singly nicked DNA, mediated formation of the oncogenic NPM–ALK translocation, which was also dependent on canonical NHEJ. Importantly, loss of DNA ligase III (LIG3), which promotes alternative NHEJ and murine translocations, did not impair the translocation frequency or alter translocation junction characteristics in human cells. Moreover, suppression of the alternative NHEJ DNA resection factor, retinoblastoma binding protein 8 (also known as CtIP), decreased translocation frequency in human cells only when canonical NHEJ activity was also reduced. Together, these results indicate that the canonical NHEJ pathway predominantly drives chromosomal translocations in human cells and highlights a species-specific difference in chromosomal rearrangement mechanisms.
- ©2014 American Association for Cancer Research.
Volume 4, Issue 11
