Figure 7.

KDM6A SUMOylation increases KDM6A–SND1 interaction and involved in the stability and restart of DNA replication forks. (A) SUMO1 and SAE1 were identified by mass spectrometry in KDM6A coimmunoprecipitation samples. (B, C) Detection of KDM6A SUMOylation by Co-IP followed by western blot with indicated antibodies. Co-IP was performed in HEK293T cells transfected with either Flag-SUMO1 alone (B) or together with KDM6A plasmids (C). SUMOylation of endogenous (B) and exogenous KDM6A (C). (D) The co-localization between KDM6A and SUMO1 in cells. Representative images with scale bars (5 μm) and quantifications are shown. (E) SUMOylation of KDM6A mutants. co-IP was performed in HEK293T cells transfected with Flag-SUMO1 and KDM6A plasmids and followed by western blot with indicated antibodies. (F) The interaction between KDM6A mutant(s) and SND1. Co-IP was performed in HEK293T cells transfected with the plasmid of HA-KDM6A and Myc-SND1, followed by western blot with indicated specific antibodies against tag. (G) Complement of KDM6A^K90A partially restore the colony formation (left) and survival (right) of HeLa cells lacking endogenous KDM6A protein in response to CPT. (H) The enrichment of SND1, KDM6A^K90A, KDM6A^wt on nascent DNA was determined by iPOND and western blot. (I) The histogram represented the restoration in length of DNA fiber in HeLa cells lacking endogenous KDM6A with KDM6A^K90A and KDM6A^wt complement respectively. All experiments were independently repeated three times, *P < 0.05.