Fig. 3: Regulation of EZH2 degradation by DCAF1-mediated phosphorylation.
a Control (Ctrl) and DCAF1-depleted SW620 colon cancer cells were treated with MG132 (10 µM) for 12 h, and cell lysates were collected and immunoblotted with antibodies against EZH2, DCAF1, EZH2T367p, and H3K27me3 (left panel). EZH2-depleted SW620 cells were infected with lentiviruses expressing 3×FLAG-EZH2T367A phospho-blocking mutant and T367D phospho-mimicking mutant followed by treatment with MG132 (10 µM) for 12 h and analyzed (right panel). Actin was used as a loading control. Data are representative of three independent experiments. b SW620 cells used in (A) were treated with CHX (50 µg/ml) for 6 h, and cell lysates were prepared and analyzed by Western blotting. Data are representative of three independent experiments. c Circular dichroism spectra of wild type EZH2 (300-450) and EZH2T367D (300-450) were recorded at protein concentrations of 5 µM in spectrum buffer (20 mM K2HPO4/KH2PO4, pH 7.4, 25 mM KCl, and 100 µM TCEP) at 25 °C using a JASCO J-810 spectropolarimeter. The observed ellipticity in millidegrees, θ, was converted into the mean residue ellipticity, θMRW. d His-tagged EZH2 wild type, T367A mutant, and T367D mutant were immobilized on Ni-NTA-agarose and incubated with FLAG-SUZ12 and FLAG-EED. After extensive washing, bound SUZ12 and EED proteins were fractionated by 10% SDS-PAGE, and probed with anti-FLAG antibody. Input lanes represent 20% of SUZ12 and EED used in the binding reactions. Data are representative of three independent experiments. e HA-EZH2 wild type, T367A mutant, and T367D mutant were expressed in SW620 cells and immunoprecipitated using anti-HA antibody. The binding of endogenous EED and SUZ12 to HA-EZH2 was analyzed by Western blotting. Data are representative of three independent experiments. (a, b, d, e) Source data are provided as a Source Data file.
