Fig. 1: G9a is SUMO-modified.

a Domain structure of G9a depicting the glutamine (E)- and cysteine (Cys)-rich regions, nuclear localization signal (NLS), ankyrin repeats (Ank), and the catalytic (SET) domain (upper panel). Numbers indicate amino acid residues. Arrowheads indicate potential sumoylation sites at lysine (K) 79 (K79), K152, K256, and K799. Alignment of the G9a complementary DNA (cDNA) sequence from humans (Gene ID: 10919), mouse (Gene ID: 110147), and rat (Gene ID: 361798) revealed a high degree of conservation of the putative SUMOylation sites (boxed). b HEK293T cells were transfected with constructs encoding Flag-G9a, SUMO1, and sentrin-specific protease 1 (SENP1) as indicated. Lysates were subjected to immunoprecipitation with Flag-agarose beads, followed by immunoblotting with anti-SUMO1 antibody. Anti-Flag antibody was used to detect the expression of G9a. β-Actin served as a loading control. Arrow indicates SUMOylated G9a. Arrowhead indicates a non-specific band. c C2C12 cells were transfected with pCS2 empty vector (−) or with a SUMO1 plasmid (+). Endogenous G9a was immunoprecipitated using anti-G9a antibody, followed by western blotting with anti-SUMO1 antibody. Immunoglobulin G (IgG) was used as a negative control. Ten percent of lysate was loaded to detect G9a expression (Input). β-Actin was used as a loading control. d HEK293T cells were transfected with constructs encoding wild-type Flag-G9a, and each of the single mutants, Flag-G9aK79R, Flag-G9aK152R, Flag-G9aK256R, Flag-G9aK799R, and SUMO1, as indicated. Lysates were immunoprecipitated with Flag-agarose beads, followed by western blotting with anti-SUMO1 antibody. e C2C12 myoblasts were transfected with Flag-G9a or the single mutants, Flag-G9aK79R, Flag-G9aK152R, Flag-G9aK2562R, and Flag-G9aK799R, in the presence of SUMO1. Nuclear extracts were used to perform a calorimetric global protein SUMOylation assay. Cells transfected with only SUMO1 were used as a negative control. Percentage SUMOylation for each mutant compared to wild-type G9a is shown. f Cells were transfected with Flag-G9a or Flag-G9a4KR in the absence or presence of SUMO1 as indicated. Lysates were immunoprecipitated with Flag-agarose beads, followed by western blotting with anti-SUMO1 antibody. g COS-7 cells were transfected with wild-type Flag-G9a and each of the single mutants, Flag-G9aK79R, Flag-G9aK152R, Flag-G9aK256R, and Flag-G9aK799R. Cells were fixed and stained with anti-Flag (red) antibody. Nuclei were stained and visualized with 4′,6-diamidino-2-phenylindole (DAPI) (blue). h COS-7 cells were transfected with Flag-G9a and Flag-G9a4KR in the absence or presence of SUMO1. Cells were fixed and stained with anti-Flag (red) antibody. Nuclei were stained and visualized with DAPI (blue). Scale bar = 100 μm. Significance was determined using Student’s t-test (*p<0.05)