<i>O</i>-GlcNAcylation regulates the stability and enzymatic activity of the histone methyltransferase EZH2.

Abstract

Protein <i>O</i>-glycosylation by attachment of β-<i>N</i>-acetylglucosamine (GlcNAc) to the Ser or Thr residue is a major posttranslational glycosylation event and is often associated with protein folding, stability, and activity. The methylation of histone H3 at Lys-27 catalyzed by the methyltransferase EZH2 was known to suppress gene expression and cancer development, and we previously reported that the <i>O</i>-GlcNAcylation of EZH2 at S76 stabilized EZH2 and facilitated the formation of H3K27me3 to inhibit tumor suppression. In this study, we employed a fluorescence-based method of sugar labeling combined with mass spectrometry to investigate EZH2 glycosylation and identified five <i>O</i>-GlcNAcylation sites. We also find that mutation of one or more of the <i>O</i>-GlcNAcylation sites S73A, S76A, S84A, and T313A in the N-terminal region decreases the stability of EZH2, but does not affect its association with the PRC2 components SUZ12 and EED. Mutation of the C-terminal <i>O</i>-GlcNAcylation site (S729A) in the catalytic domain of EZH2 abolishes the di- and trimethylation activities, but not the monomethylation of H3K27, nor the integrity of the PRC2/EZH2 core complex. Our results show the effect of individual <i>O</i>-GlcNAcylation sites on the function of EZH2 and suggest an alternative approach to tumor suppression through selective inhibition of EZH2 <i>O</i>-GlcNAcylation.