The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells.

Clastogen exposure can result in chromosomal rearrangements, including large deletions and inversions that are associated with cancer development. To examine such rearrangements in human cells, here we developed a reporter assay based on endogenous genes on chromosome 12. Using the RNA-guided nuclease Cas9, we induced two DNA double-strand breaks, one each in the GAPDH and CD4 genes, that caused a deletion rearrangement leading to CD4 expression from the GAPDH promoter. We observed that this GAPDH-CD4 deletion rearrangement activates CD4+ cells that can be readily detected by flow cytometry. Similarly, double-strand breaks in the LPCAT3 and CD4 genes induced an LPCAT3-CD4 inversion rearrangement resulting in CD4 expression. Studying the GAPDH-CD4 deletion rearrangement in multiple cell lines, we found that the canonical non-homologous end joining (C-NHEJ) factor XLF promotes these rearrangements. Junction analysis uncovered that the relative contribution of C-NHEJ appears lower in U2OS than in HEK293 and A549 cells. Furthermore, an ATM kinase inhibitor increased C-NHEJ-mediated rearrangements only in U2OS cells. We also found that an XLF residue that is critical for an interaction with the C-NHEJ factor X-ray repair cross-complementing 4 (XRCC4), and XRCC4 itself are each important for promoting both this deletion rearrangement and end joining without insertion/deletion mutations. In summary, a reporter assay based on endogenous genes on chromosome 12 reveals that XLF-dependent C-NHEJ promotes deletion rearrangements in human cells and that cell type-specific differences in the contribution of C-NHEJ and ATM kinase inhibition influence these rearrangements.