Nuclear compression-mediated DNA damage drives ATR-dependent Lamin expression and mouse ESC differentiation

Embryonic stem cells (ESCs) which are susceptible to DNA damage depend on a robust and highly efficient DNA damage response (DDR) mechanism for their survival. However, the implications of physical force-mediated DNA damage on ESC fate remains unclear. Here we assessed the importance of DNA damage in ESC differentiation by culturing mouse ESCs (mESCs) on substrates of varying stiffness, which has been shown to induce stem cell differentiation. We show that stiffness-dependent mESC spreading induces DNA damage through nuclear compression that leads to loss of pluripotency, expression of Lamin A/C and germline markers. DNA damage and induction of Lamin A/C are observed in the presence of differentiating agents, as well as during mESC differentiation on cell derived matrices, identifying DNA damage as an early event in differentiation. Differentiation was also associated with reduction of DNA damage and activation of the DDR factor – ATR. While ATR is typically known to play roles in DDR pathway, its role during stiffness-mediated nuclear compression and mESC differentiation is unknown. Nuclear enrichment of activated ATR on stiff substrates and reduction of Lamin A/C expression due to ATR inhibition suggests that mESC differentiation is driven by nuclear compression-mediated DNA damage and involves ATR-dependent modulation of Lamin A/C. Overall design: To probe the effect of stiffness mediated mechanoadaptation on Tg2A mouse embryonic stem cells (mESCs), we cultured mESCs on gels of varying stiffnesses (0.6 kpa, 4 kPa and 33 kpa) coated with fibronectin. RNA isolation of whole cell lysates was done at different timepoints (3hrs, 6 hrs, 24 hsr and 72 hrs) and compared with a pluripotent control which was cultured on gelatin coated dish in presence of LIF. A DNA damage positive control was used by treating the cells with etoposide (ETO) on 0.6 kPa for 24 hrs. Samples were pooled from 3 independent experiments and run as 2 replicates.