O-GlcNAcylation regulates EZH2 protein stability and function

Abstract

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.

Fig. 1.

OGT knockdown reduces H3 trimethylation at K27. (A) OGT depletion decreases the level of H3K27me3 but not other H3 methylation products. Total cell lysates or histones purified from MCF7 cells mock transfected (NT), transfected with scramble RNA (Scr), or two different siOGT (#1 and #2) separately or together were subjected to Western blot using indicated Abs. (B) OGT knockdown decreases global H3K27me3 measured by mass spectrometry. Histones purified from MCF7 cells transfected with indicated siRNAs were subjected to SDS/PAGE, followed by LC/MS analysis.

Fig. 2.

OGT knockdown reduces EZH2 protein stability. (A) OGT knockdown decreases the protein level of EZH2 but not UTX or JMJD3. (B) Reduction of EZH2 by OGT knockdown can be rescued by adding back resistant OGT. (C) OGT knockdown decreases the protein levels of PRC2 components. Total lysates or histone extracts from MCF7 cells transfected with scramble siRNA (Scr) or siOGT were subjected to Western blot using indicated Abs. For B, cells were treated with scramble (Scr) siRNA or siOGT for 24 h, followed by transfection of vector alone (lanes 1 and 2) or the plasmid encoding the siRNA-resistant OGT (lane 3) for 2 d. (D) OGT knockdown destabilizes EZH2. MCF7 cells were transfected with scramble siRNA (Scr) or siOGT for 3 d and subsequently treated with cycloheximide at the final concentration of 50 μg/mL and harvested at the indicated time points for Western blot. Band intensities were measured by ImageJ. Normalization was done by dividing the EZH2 signal to α-tubulin signal. P values were measured by Student’s t test. The results are presented as mean ± SD. *P = 0.031, n = 3.

Fig. 3.

OGT stably interacts with EZH2 in the PRC2 complex. (A) Exogenous OGT associates with exogenous EZH2. Total lysates from 293T cells expressing OGT-V5 and/or EZH2-FLAG were subjected to IP with FLAG Ab (Left) or V5 Ab (Right), followed by Western blot using indicated Abs. (B) Endogenous OGT interacts with endogenous EZH2. Nuclear extracts from MCF7 cells were subjected to IP, followed by Western blot using indicated Abs. Asterisk indicates a nonspecific band. (C) OGT cofractions with PRC2 complex. Nuclear extracts from MCF7 cells were subjected to 10–50% glycerol sizing gradient sedimentation. Fractions were collected, precipitated by trichloroacetic acid, and analyzed by Western blot using indicated Abs.

Fig. 4.

S75 O-GlcNAcylation of EZH2 is required for EZH2 stability. (A) Accumulation of global O-GlcNAcylation level increases EZH2 and global H3K27me3. Total lysates or histones from MCF7 treated with DMSO or PUGNAc for 16 h were subjected to Western blot using indicated Abs. (B) Endogenous EZH2 is O-GlcNAcylated. Nuclear extracts from MCF7 cells with or without PUGNAc were subjected to Western blot with O-GlcNAc Ab or IP with IgG or EZH2, followed by Western blot using O-GlcNAc or EZH2 Ab. (C) Detection of EZH2-O-GlcNAz-FLAG. Nuclear extracts from MCF7 cells treated with O-GlcNAz for 16 h were incubated with phosphine-FLAG overnight at 4 °C. Subsequently lysates were subjected to IP in denatured condition, followed by Western blot using indicated Abs. Arrowheads indicate the band of EZH2. Asterisk indicates a nonspecific band. (D) EZH2 is O-GlcNAcylated at serine 75. An MS/MS spectrum was generated from microliquid chromatography/tandem MS. (E) Serine 75 mutation reduces EZH2 protein stability. 293T cells expressing EZH2-WT-FLAG or EZH2-S75A-FLAG were treated with cycloheximide at the final concentration of 50 μg/mL and harvested at indicated time points for Western blot using indicated Abs. Band intensities were measured by ImageJ. Normalization was done by dividing the FLAG signal to β-tubulin signal. P values were measured by Student’s t test. The results in E are presented as mean ± SD. *P < 0.05, n = 3.

Fig. 5.

The OGT-EZH2 axis suppresses specific tumor suppressor gene expression. mRNA levels of 16 genes corepressed by OGT and EZH2 were confirmed with RT-PCR in MCF7 depleted of OGT (A) or EZH2 (B). (C) OGT occupancy at the promoter regions of the 16 genes in A and B. MCF7 cells were subjected to ChIP with control IgG (white bar) or OGT Ab (black bar). (D and E) EZH2 occupancy (D) and H3K27me3 (E) associated with the promoter regions of the 16 genes in A and B depend on OGT. MCF7 cells with scramble siRNA (white bar) or siOGT (black bar) were subjected to ChIP with control IgG, EZH2, or H3K27me3 Ab. In D and E the data are shown as fold enrichment relative to IgG. GAPDH gene was used as a negative control. All results from A to E are presented as mean ± SD. P values were measured by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, n = 3.