O-GlcNAcylation regulates the stability and enzymatic activity of the histone methyltransferase EZH2.

Protein O-glycosylation by attachment of ß-N-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 O-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 O-GlcNAcylation sites. We also find that mutation of one or more of the O-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 O-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 O-GlcNAcylation sites on the function of EZH2 and suggest an alternative approach to tumor suppression through selective inhibition of EZH2 O-GlcNAcylation.

Influenza A surface glycosylation and vaccine design.

We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAV-specific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.

O-GlcNAcylation regulates EZH2 protein stability and function.

O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) is the only known enzyme that catalyzes the O-GlcNAcylation of proteins at the Ser or Thr side chain hydroxyl group. OGT participates in transcriptional and epigenetic regulation, and dysregulation of OGT has been implicated in diseases such as cancer. However, the underlying mechanism is largely unknown. Here we show that OGT is required for the trimethylation of histone 3 at K27 to form the product H3K27me3, a process catalyzed by the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the polycomb repressive complex 2 (PRC2). H3K27me3 is one of the most important histone modifications to mark the transcriptionally silenced chromatin. We found that the level of H3K27me3, but not other H3 methylation products, was greatly reduced upon OGT depletion. OGT knockdown specifically down-regulated the protein stability of EZH2, without altering the levels of H3K27 demethylases UTX and JMJD3, and disrupted the integrity of the PRC2 complex. Furthermore, the interaction of OGT and EZH2/PRC2 was detected by coimmunoprecipitation and cosedimentation experiments. Importantly, we identified that serine 75 is the site for EZH2 O-GlcNAcylation, and the EZH2 mutant S75A exhibited reduction in stability. Finally, microarray and ChIP analysis have characterized a specific subset of potential tumor suppressor genes subject to repression via the OGT-EZH2 axis. Together these results indicate that OGT-mediated O-GlcNAcylation at S75 stabilizes EZH2 and hence facilitates the formation of H3K27me3. The study not only uncovers a functional posttranslational modification of EZH2 but also reveals a unique epigenetic role of OGT in regulating histone methylation.

Protein kinase A-mediated serine 35 phosphorylation dissociates histone H1.4 from mitotic chromosome.

Global histone H1 phosphorylation correlates with cell cycle progression. However, the function of site-specific H1 variant phosphorylation remains unclear. Our mass spectrometry analysis revealed a novel N-terminal phosphorylation of the major H1 variant H1.4 at serine 35 (H1.4S35ph), which accumulates at mitosis immediately after H3 phosphorylation at serine 10. Protein kinase A (PKA) was found to be a kinase for H1.4S35. Importantly, Ser-35-phosphorylated H1.4 dissociates from mitotic chromatin. Moreover, H1.4S35A substitution mutant cannot efficiently rescue the mitotic defect following H1.4 depletion, and inhibition of PKA activity increases the mitotic chromatin compaction depending on H1.4. Our results not only indicate that PKA-mediated H1.4S35 phosphorylation dissociates H1.4 from mitotic chromatin but also suggest that this phosphorylation is necessary for specific mitotic functions.