Figure 2. The EZH2_CXC-DNA interaction interface is critical for H3K27 methylation on nucleosomes.

(A) Binding reactions with indicated concentrations of PRC2 (lanes 1–10), PRC2^CXC>A (lanes 11–20), PRC2^CXC>A/EED>A (lanes 21–30), or PRC2^EED>A (lanes 31–40) and 45 nM 6-carboxyfluorescein-labeled mononucleosomes, analyzed by EMSA on 1.2% agarose gels. The EMSA with PRC2^EED>A was run on a separate gel. (B) Quantitative analysis of EMSA data in A by densitometry of 6-carboxyfluorescein signals from independent experiments (n = 3); error bars, SEM. (C) Western Blot (WB) analysis of H3K27me1 and H3K27me3 formation in HMTase reactions with indicated concentrations of PRC2 and PRC2^CXC>A on 446 nM mononucleosomes (lanes 1–7) or 223 nM dinucleosomes (lanes 8–14). Note that these concentrations result in equal numbers of nucleosomes and therefore equal numbers of H3 substrate molecules in the reactions on mono- and dinucleosomes, as can be seen from the Coomassie-stained gel of the reactions in Figure 2—figure supplement 1B. H4 WB signal served as control for western blot processing.

Figure 2—figure supplement 1. The EZH2_CXC-DNA interaction interface is critical for H3K27 methylation on nucleosomes (related to Figure 2).

(A) Coomassie-stained 4–12% SDS-PAGE of the HMTase reactions shown in Figure 2C. Xenopus laevis (X.l.) nucleosomes were used for these experiments. The short 5 kDa PHF_C fragment is not visible on this gel. (B) Left: Mass spectrometry-based HMTase assays monitoring H3K27me1 formation by PRC2 (blue; upper two panels) and PRC2^CXC>A (green; lower two panels) on H3_18-42 peptides. Representative full ESI MS Spectra (top part) and deconvoluted spectra (lower part) are shown for the individual runs. For both runs, peaks indicated by the brackets were used for quantification of H3K27me1 formation. Right: Symbols represent percentages of peptides carrying H3K27me1 in technical triplicate experiments; error bars show SD.