Pluripotent cells rapidly proliferate while robustly protecting their genome integrity. Notably, mouse embryonic cells (ESCs) within the inner cell mass of blastocyst stage embryos have an average cell cycle time of ten hours, while pluripotent epiblast cells (EpiSCs) from gastrulating embryos complete the cell cycle within three hours1. This implies that EpiSCs possess a superior ability to replicate their DNA under extraordinary conditions.
We reveal that compared to ESCs, EpiSCs have an enhanced capacity to manage Hydroxyurea (HU) induced DNA replication stress. Western blotting revealed elevated basal and HU induced activity of ATR, the master regulator of the replication stress response, in EpiSC as compared to ESC cultures. Pulsing with thymidine analogues revealed that both EpiSCs and ESCs experience reduced DNA synthesis following HU treatment, but that unlike ESCs, EpiSCs adapt within hours to resume DNA replication. Flow cytometry, western blotting, and live-cell imaging further revealed that in sharp contrast to ESCs, replication stressed EpiSCs experience minimal DNA damage and avoid apoptosis. We have previously identified that replication stress induces cell death primarily during mitosis through apoptotic pathways2. In agreement, while replication stressed ESCs progress into mitosis and apoptose under replication stress, mitotic entry in HU treated EpiSCs is prevented in an ATR-dependent manner. To elucidate differential signalling in the ESC versus EpiSC replication stress response we performed phosphoproteomic analysis on both cell types treated for eight hours with Hydroxyurea ± ATR inhibition. Identification and unsupervised clustering of 16,197 unique phospho-peptides revealed AKT and ATM/ATR substrates as some of the most significantly different phospho-peptides between replication stressed ESCs and EpiSCs cultures.
Collectively, our work reveals a previously uncharacterised enhanced ability of pluripotent EpiSCs to sense and respond to replication stress in an ATR-dependent manner. This enhanced capability prevents apoptosis in EpiSCs and enables DNA synthesis to continue during unfavourable conditions.