Fig. 2

ATF7IP is tri-methylated by G9a/GLP in mESCs. a–f 3xFLAG-tagged ATF7IP was immunopurified from mESC lines and subjected to LC–MS/MS analysis or western blot analysis. a A tandem mass (MS/MS) spectrum of the tri-methylated “DSVEEPQKKVFKARKTMRAS” peptide from wild-type (WT) mESCs expressing 3xFLAG-ATF7IP. The detected y ions (pink) indicated tri-methylation occurred on the H3K9-like lysine (K16, pink). b tri-methylation status of exogenous ATF7IP in mESC lines mutant for G9a, Glp, or both. After an immunoprecipitation with FLAG M2 beads, western blot analysis was done with indicated antibodies. WT and KR indicate wild-type and K16R mutant of ATF7IP, respectively. c Quantification from the blots in (b) is shown. Error bars indicate ± SEM. d Percentage of methylation states of the ATF7IP K16 in WT or G9a/Glp DKO mESCs, as estimated by MS chromatogram. ND, not detected. e methylation status of exogenous ATF7IP was not affected by depletion of SETDB1/ESET. After serial treatment of tamoxifen (OHT), 3xFLAG-tagged ATF7IP was immunoprecipitated by FLAG M2 beads. Western blot analysis was done with indicated antibodies. Efficient knock-out in the tamoxifen-treated cell population was confirmed by the blot with anti-SETDB1 antibody. f Methylation status of exogenous ATF7IP in Suv39h1/h2 DKO mESCs was comparable to that in WT cells