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 glycosylation with β-N-acetyl-D-glucosamine (O-GlcNAcylation) is a widespread and dynamic modification in both cytosol and nucleus (1, 2). It occurs by O-linked N-acetylglucosamine (GlcNAc) transferase (OGT)-catalyzed glycosylation at the hydroxyl group of serine or threonine residue of the protein substrate, and removal of the O-GlcNAc group is catalyzed by the glycosidase O-GlcNAcase (OGA) (3⇓⇓–6). How protein O-GlcNAcylation exerts its effect is largely unknown, but previous studies show that it could induce a conformational change to initiate protein folding (7), compete with phosphorylation at the same or proximal serine or threonine (8), disrupt protein–protein interaction (9), serve as a protein recruiting signal (10), or regulate protein stability (11).
More than thousands of proteins are modified by O-GlcNAcylation. These proteins are involved in a variety of biological and pathological processes, including epigenetic regulation, transcription, translation, signal transduction, cell division, synaptic plasticity, embryonic stem cell identity, type II diabetes, Alzheimer’s disease, and tumor malignancy (8, 12⇓⇓–15). O-GlcNAcylation regulates transcription and epigenetics at least through the following two mechanisms. First, OGT adds GlcNAc to DNA-binding transcription factors, such as p53, c-Myc, etc. (16), and to histone proteins such as histone H2A at T101, H2B at S36 and S112, H3 at S10 and T32, and H4 at S47 (17⇓⇓–20). O-GlcNAcylation of H2B at S112 facilitates the ubiquitination of K120, leading to up-regulation of transcriptional elongation (18). The functions of most histone O-GlcNAcylations are currently not clear, nor do we completely understand how OGT is recruited to chromatin, although some progress has been made. It is known that OGT can bind to corepressor mSin3A (21) and also associate with the DNA demethylase TET family proteins TET2 and TET3 and potentiate TET family protein-mediated gene activation (22⇓⇓–25). Another critical mediator of OGT is the polycomb repressive complex 2 (PRC2).
PRC2 is a conserved complex that in humans contains the enhancer of zeste homolog 2 (EZH2), SUZ12, EED, and RbAp46/48 (26, 27). As a Su(var)3-9, enhancer of zeste and trithorax domain-containing enzyme, the major function of EZH2 is to catalyze the transfer of methyl groups to the K27 residue of histone H3 to form H3K27me3 and to induce a signal to recruit polycomb repressive complex 1 (PRC1) for establishing the silenced chromatin (26⇓⇓–29). EZH2/PRC2 has been shown to play critical roles in diverse biological processes, such as development, stem cell maintenance, and X-chromosome inactivation (30). Most importantly, EZH2 overexpression in various types of cancers has been linked to oncogenesis, partly via H3K27me3 in promoters of specific tumor suppressor genes, and thus causes gene silencing (31). Targeting EZH2 is believed to be a promising strategy for cancer therapy (32, 33).
Functional association of OGT and EZH2/PRC2 was first reported in Drosophila. Sxc/Ogt, the Drosophila homolog of mammalian OGT, was identified as a polycomb group (PcG) protein involving in polycomb repression during larvae development (34, 35). The chromosomal location of O-GlcNAc coincides with the PcG response elements in both Drosophila (34, 35) and Caenorhabditis elegans (36). Interestingly, mutations in PRC2 subunits decrease the level of Ogt protein and global O-GlcNAcylation in mouse embryonic stem cells (37). These observations indicate that OGT and PRC2 may function dependently on each other. Nevertheless, it is obscure how OGT regulates PcG repression. Three of the PRC1 components, Ph, Pc, and Ring, are likely Sxc/Ogt substrates in Drosophila (34). However, these observations have not been confirmed by mass spectrometry analysis, and no function has been reported with regard to the O-GlcNAcylation of these proteins (34). In the present study, using an unbiased small-scale screening, we independently demonstrate that OGT depletion leads to the down-regulation of H3K27me3. We also found that OGT is able to associate with EZH2 and the PRC2 complex and that EZH2 is O-GlcNAcylated at S75 to maintain its stability and activity.