HMTase activity assay

The HMTase activity of PRC2 was measured using Perkin Elmer AlphaLISA kits [56]. Enzymatic reactions were promoted by incubating 10 nM PRC2 with assay buffer (50 mM Tris-HCl, pH 8.0; 50 mM NaCl; 1 mM dithiothreitol; 0.01% Tween-20; and 0.01% bovine serum albumin) containing 100 nM biotinylated histone H3 (21–32) peptide substrate and 5 μM SAM for 120 min at room temperature. Anti-trimethyl-histone H3 lysine 27 (anti-H3K27me3) AlphaLISA Acceptor beads and Streptavidin Donor beads (0.08 mg/mL) were preincubated for 1 h in 1 × Epigenetics Buffer 1. The presence of the histone H3 product methyl marker was detected by the addition of the preincubated AlphaLISA beads (final concentration of 5 μg/mL). Activity was allowed to proceed for 1 h at room temperature. The assay plates were subsequently analyzed in a Perkin Elmer Envision plate reader using the AlphaLISA setting for optimal signal detection with a 615 nm filter after sample excitation at 680 nm. The emission signal at 615 nm was used to quantify HMTase activity. The activity of PRC2 in the absence of C10ORF12 was defined as the relative activity value of 1.0. The data are expressed as the means ± standard errors of the means (SEMs) of at least three independent experiments. Statistical analysis of the differences between PRC2 and PRC2 with C10ORF12FL or C10ORF12 (619–989) was conducted using GraphPad Prism 7.0. The significance of the differences was assessed using Student’s t test (P < 0.05 was considered statistically significant).