Supplementary Figure 4: Stimulatory peptides relieve RNA-mediated inhibition of PRC2.

a,b, HMTase assays were carried out in the presence of 0.5 μM wild-type (WT) or mutant 1 (mt1) PRC2 or PRC2-AEBP2, 2.0 μM nucleosome substrate, in the presence (+) or absence (-) of 80 μM H3K27me3 peptide and in the presence (+) or absence (-) of 4.0 μM G4 256 RNA. Representative Coomassie blue-stained SDS–PAGE (top) and the corresponding radiograms (middle) are presented, with bar plots (bottom) representing the HMTase activities quantified based on three replicates. The data represented by black bars in panels (c) and (d) were used to generate Fig. 4a. e,f, Representative Coomassie blue-stained SDS–PAGE and the corresponding radiograms used for quantifying the HMTase activities presented in Fig. 4c. f, HMTase assays were carried out in the presence of 0.5 μM PRC2-AEBP2, 2.0 μM nucleosome substrate and G4 24 RNA (e) or G4 256 RNA (f) at concentrations of either 0, 4 or 8 μM and stimulatory peptides, as indicated. g, Fluorescence anisotropy used to quantify the affinity of PRC2-AEBP2 to G4 24 RNA in the presence or absence of 10 μM of the EED inhibitor A395 or the negative control A395N. Error bars represent standard deviation based on three independent experiments that were performed on different days. h, Resulting dissociation constants (Kd), Hill coefficients and the derived ΔΔG values are indicated. Standard errors are indicated. i, The coordinates of A395, as previously identified by X-ray crystallography (PDB: 5K0M, He, Y. et al., Nat Chem Biol. 13, 389–395, 2017), are presented on the high-resolution cryo-EM structure of PRC2 (PDB: 6C23) by superimposing EED from both structures. Orange and red spots represent RNA-linked polypeptides that were identified in 2 or 3 independent RBDmap experiments respectively.