Supplementary Figure 4: The PRC2 catalytic core binds G4 RNA and this blocks it interaction with the nucleosome core particle.
From: G-tract RNA removes Polycomb repressive complex 2 from genes
(a) Fluorescence anisotropy measuring binding of the PRC2 catalytic core (EZH2, EED, SUZ12 VEFS domain) directly to fluorescein-labeled [G4A4]4 RNA in nucleosome binding buffer (40 mM KCl). Mean and S.E., n=3. (b) As (a), except for the G4-forming 24 nt sequence within PIM1 RNA. (c) Fluorescence intensity measuring binding of the PRC2 catalytic core directly to MDCC-labeled wild type core nucleosome particles (reconstituted with 147 bp DNA) in the presence or absence of competing 500 nM [G4A4]4 RNA (mean and S.E., n=3). (d) Fluorescence anisotropy measuring binding of the PRC2 catalytic core to fluorescein-labeled [G4A4]4 RNA in the presence of unlabeled PIM1 G4 RNA or an unlabeled non-G4-forming part of PIM1 RNA. Mean and S.E., n=3. (e) Immunoblotting for SUZ12, JARID2, HMGN1 and H3 after co-immunoprecipitation of PRC2 from Jarid2GT/GT or matched wild-type E14 ESC with nucleosomes containing HA-tagged histone H2A (reconstituted with either 185 bp or 147 bp DNA) from mock or RNaseA-treated nuclear extract. Representative of 2 independent experiments. (f) Immunoblotting for SUZ12, AEBP2, HMGN1 and H3 after co-immunoprecipitation of PRC2 from Aebp2GT/GT or paired WT ESC with nucleosomes containing HA-tagged histone H2A (reconstituted with either 185 bp or 147 bp DNA) from mock or RNaseA-treated nuclear extract. Representative of 2 independent experiments.
