Effect of T350 phosphorylation on gene repression activity of EZH2 in BJ cells

The Polycomb group (PcG) protein enhancer of zeste homolog 2 (EZH2) plays an essential role in histone methylation-mediated epigenetic gene silencing. We demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and 2 (CDK2) bind to and phosphorylate EZH2 at threonine 350 (T350) in an evolutionally conserved motif. T350 phosphorylation is required for EZH2 function in silencing of genes that promote differentiation and inhibit proliferation and migration. Blockage of T350 phosphorylation by generating a T350A mutation largely diminishes the global effect of EZH2 on gene silencing. Four experiments have been designed. BJ cells were transfected with si-Control, si-EZH2, si-EZH2 plus RNAi resistant lentivirus expressed EZH2, or si-EZH2 plus RNAi resistant lentivirus expressed T350A mutant for 60 hours. Each experiment was triplicated.