DNA damage response in single RPE-FUCCI cells

E2F transcription factors control the expression of a large network of cell cycle genes and are essential for S-phase entry. Cancers often demonstrate upregulation of E2F target gene expression, which can be partially explained by loss of the G1/S checkpoint and increased percentages of replicating cells. However, we now demonstrate that cycling individual neoplastic cells can display abnormally high levels of E2F-dependent transcription using single cell RNA sequencing. To test how this affects their DNA damage response, we deleted the atypical E2F transcriptional repressors (E2F7/8) in untransformed cells. This intervention specifically increased the expression of E2F target genes during S and G2-phase without overriding the G1/S-checkpoint. Live cell imaging revealed that cells in S-G2 with deregulated E2F activity failed to arrest and underwent unscheduled mitosis after neocarzinostatin-induced DNA damage. In contrast, cells with physiological E2F-activity completed S-phase and then activated the APC/C-Cdh1 via repression of the E2F-target Emi1, leading to a G1-like arrest. Interestingly, ~30% of these 4N-G1 cells could eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, resulting in the formation of tetraploid cells. Cells with deregulated E2F activity fail to exit the cell cycle after DNA damage and likely acquire more genetic changes. The observed elevated E2F-dependent transcription in cancer cells could therefore potentially promote malignant progression and reduce sensitivity to anti-cancer drugs. Retina-pigment epithelial (RPE-hTERT) cells with stable expression of the FUCCI system were treated with 200 ng/mL neocarzinostatin or vehicle, and harvested after 24 and 48 hours. We compared E2F7/E2F8 double knockout cells with wild type cells. E2F7 and 8 were deleted using CRISPR/Cas9. Prior to single cell capturing, we either labeled E2F/8 mutant or wild type cells with the lipophilic dye DiD, and mixed the labeled and unlabeled cells prior to single cell capturing. Using fluorescence microscopy immediately prior to cell capturing and lysis we could determine the genotype of each individual cell.