SUZ12 and EED mutations abrogateHMTactivity. (A) In vitro HMT assay of baculovirus-expressed components demonstrating loss of activity with SUZ12 and EED mutant complexes (top panels) compared with WT controls. The SUZ12 mutants F603L, D605V, and E610G and the EED mutant G255D were identified in this study. Sf9 indicates uninfected insect cells. The bottom panel (Coomassie-stained histones) confirms the presence of the histone substrates in each reaction. (B) Coomassie-stained SDS-PAGE of immunoprecipitated PRC2 components Ezh2, Suz12, and Eed confirming the presence of each of these components in the assay. (C) Protein blots of immunoprecipitated PRC complexes with Abs indicated further confirming the presence of the 3 PRC2 components. All results were obtained in 2 independent clones, however, only clone 1 is shown here. (D) Normalized HMT assay results. The intensity of the H3K27me3 bands was normalized against the protein blot bands (Suz12 bands for Suz12 mutants and Eed bands for the EED mutant) and are shown as a percentage of WT activity. The error bars (SDs from mean values) are derived from 2 separate experiments with 2 independently derived clones.