In vitro histone methyltransferase assay

In each 10-µL reaction, recombinant PRC2-5m, H3 (New England Biolabs, M2503S), and S-[methyl-¹⁴C]-adenosylmethionine (PerkinElmer, NEC363050UC) were mixed in methylation buffer (50 mM Tris-HCl at pH 8.0 at 30°C, 100 mM KCl, 2.5 mM MgCl₂, 0.1 mM ZnCl₂, 2 mM 2-mercaptoethanol, 0.1 mg/mL bovine serum albumin, 5% [v/v] glycerol). All of the methylation reactions were incubated for 1 h at 30°C followed by addition of 4× loading dye to stop each reaction and heated for 5 min at 95°C. Each reaction was then loaded onto a 4%–12% Bis-Tris gel (Thermo Fisher, NP0322BOX). Gel electrophoresis was carried out for 48 min at room temperature at 180 V. Gels were stained by InstantBlue for 1 h and destained with water overnight. Three sheets of Whatman 3-mm chromatography paper were put underneath the gel, and gels were scanned followed by vacuum drying for 60 min at 80°C. Dried gels were subjected to phosphorimaging plates, and radioactive signal was acquired with a Typhoon Trio PhosphorImager (GE Healthcare). Densitometry and analysis were carried out with ImageQuant software (GE Healthcare). For activity assays on nucleosomes in Figure 5, recombinant trinuclesomes were reconstituted as described previously (Wang et al. 2017b), and native nucleosomes of HEK293 cells were purchased from Asmbio (52015); methylation buffer contained 10 mM KCl instead of 100 mM KCl, and reaction time was 4 h. For the experiments of preincubating WT PRC2 with unlabeled SAM, PRC2 was incubated with 0.3 mM SAM at the precision cleavage step of the purification process overnight at 4°C.