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. Author manuscript; available in PMC: 2014 Jul 24.
Published in final edited form as: Nat Commun. 2014;5:3127. doi: 10.1038/ncomms4127

Figure 4. The EED:PRC1 interaction alters PRC2 histone methyltransferase and PRC1 histone ubiquitin E3 ligase activities.

Figure 4

a, PRC1 proteins disassociate EED:EZH2 complexes. EZH2 protein was incubated with halo-tagged EED in the presence of different amounts of BMI1, RING1B or GUS proteins. Halo-Link PD followed by immunoblot analysis was performed. b, BMI1 and RING1B inhibit PRC2 mediated methylation of histone H3 in vitro. Indicated amounts of purified PRC2, RING1B, BMI1, and GUS were incubated with recombinant histone H3 with unmethylated H3K27 for two hours. Immunoblot analyses with anti-H3K27me3 and anti-H3K27me2 antibodies were then performed. c, BMI1 and RING1B inhibit PRC2 mediated methylation of nucleosomes in vitro. Indicated amounts of purified recombinant PRC2, RING1B, BMI1, and GUS proteins were incubated with nucleosomes containing unmethylated H3K27. ELISA with anti-H3K27me3 antibody was then performed. Assays were repeated thrice in triplicates. All error bars represent ±s.e.m. d, EED enhances RING1B mediated ubiquitination of histone H2A in vitro. Indicated amounts of purified EED, RING1B, BMI1, and GUS were incubated with recombinant histone H2A for one hour. Immunoblot analyses with anti-uH2A antibody were then performed to measure histone H2A ubiquitin E3 ligase activity. Assays were independently repeated three times. Normalized band intensity by densitometry from the three independent assays is represented as bar graphs below the immunoblot. Error bars represent ±s.e.m. e, EED enhances PRC1 mediated ubiquitination of nucleosome H2A in vitro. Purified EED, RING1B, BMI1, and GUS were incubated with reconstituted mono-nucleosomes with un- or tri-methylated H3K27. Immunoblot analyses with anti-uH2A antibody were then performed to measure histone H2A ubiquitin E3 ligase activity. Normalized band intensity by densitometry from three independent assays is represented as bar graphs blow the immunoblot. Error bars represent ±s.e.m. f, Knockdown of EED and RING1B altered H3K27me3 and uH2A levels in cells. EED- and RING1B-specific siRNA duplexes and non-targeting control were transfected into DU145 cells. Expression levels of indicated proteins were assessed by immunoblot analysis. Normalized band intensity by densitometry from three independent assays is represented as bar graphs blow the immunoblot. All error bars represent ±s.e.m.