Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2-and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs.

Pluripotent embryonic stem cells (ESCs) are characterised by their capacity to self-renew indefinitely while maintaining the potential to differentiate into all cell types of an adult organism. Both the undifferentiated and differentiated states are defined by specific gene expression programs that are regulated at the chromatin level. Here, we have analysed the contribution of the H3K27me2-and H3K27me23-specific demethylases KDM6A and KDM6B to murine ESC differentiation by employing the GSK-J4 inhibitor, which is specific for KDM6 proteins, and by targeted gene knockout (KO) and knockdown. We observe that inhibition of the H3K27 demethylase activity induces DNA damage along with activation of the DNA damage response (DDR) and cell death in differentiating but not in undifferentiated ESCs. Laser microirradiation experiments revealed that the H3K27me3 mark, but not the KDM6B protein, colocalise with γH2AX-positive sites of DNA damage in differentiating ESCs. Lack of H3K27me3 attenuates the GSK-J4-induced DDR in differentiating Eed-KO ESCs. Collectively, our findings indicate that differentiating ESCs depend on KDM6 and that the H3K27me3 demethylase activity is crucially involved in DDR and survival of differentiating ESCs.