Differential regulation of the ten-eleven translocation (TET) family of dioxygenases by O-linked β-N-acetylglucosamine transferase (OGT)

Abstract

The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.

Keywords: DNA Methylation; Glucose Metabolism; Histone Modification; O-GlcNAcylation; Protein Export; TET3.

FIGURE 1.

OGT is a major TET3-interacting protein. A, silver staining gel showing the proteins copurified with FLAG-TET3 expressed in 293T cells. The Vector lane represents the proteins purified from 293T cells transfected with the control vector. The two major bands were excised and analyzed by mass spectrometry to be TET3 and OGT, respectively. The numbers and the positions of the identified OGT peptides are listed. B and C, reciprocal IP-WB analyses showing co-IP of coexpressed TET3 and OGT. Note that FLAG-TET3 and Myc-OGT were used in B and that FLAG-OGT and Myc-TET3 were used in C. All immunoprecipitations were performed with anti-FLAG antibody. Luc, luciferase. D, TET3 interacts with OGT through its C-terminal catalytic domain. Top panel, the organization of TET3 protein and the deletion mutants used for mapping the region required for OGT interaction. Various FLAG-tagged TET3 deletion mutants and the wild-type TET3 were coexpressed with GFP-OGT in 293T cells. Immunoprecipitations were performed with anti-FLAG antibody, and WB analyses were performed with either anti-GFP or anti-FLAG, as indicated. The results of OGT interaction are summarized in the top panel. E, the optimal interaction between OGT and TET3 requires both the N-terminal and C-terminal domains of OGT. Top panel, the organization of OGT protein and the deletion mutants used for analyzing the interaction with TET3. The experiments were performed essentially as in D. The results of TET3 interaction are summarized in the top panel. NLS, nuclear localization sequence.

FIGURE 2.

OGT catalyzes TET3 O-GlcNAc modification and alters TET3 subcellular localization. A, OGT catalyzes TET3 O-GlcNAc modification in an enzymatic activity-dependent manner. FLAG-TET3 was expressed alone or together with wild-type Myc-OGT, the Myc-OGT(H508A) mutant, or the Myc-OGTΔC mutant in 293T cells. FLAG-TET3 was then immunoprecipitated from the corresponding cellular extracts and analyzed for O-GlcNAc modification by WB analysis. B, OGT catalyzes O-GlcNAc modification at multiple regions of TET3. FLAG-tagged TET3 and its various deletion mutants were coexpressed with Myc-OGT in 293T cells. The expression of various TET3 constructs was verified by WB analysis (left panel). Various TET3 proteins were immunoprecipitated using anti-FLAG antibody, and the O-GlcNAc modification was detected by WB analysis using an anti-O-GlcNAc antibody (right panel). C, OGT changes TET3 subcellular localization in an enzymatic activity-dependent manner. Myc-TET3 and FLAG-OGT were expressed either alone or together in HeLa cells, and immunofluorescence staining was performed accordingly. Note that Myc-TET3 was predominantly nuclear when expressed alone (left panel). However, Myc-TET3 was predominantly cytoplasmic when coexpressed with wild-type FLAG-OGT but not with the Myc-OGT(H508A) or Myc-OGTΔC mutants. D, OGT altered TET3 subcellular localization in 293T and NIH3T3 cells. The experiments were performed as in C.

FIGURE 3.

OGT drives TET3 out of the nucleus, possibly by promoting TET3 nuclear export. A, TET3 nuclear localization is determined by a single nuclear localization sequence. The putative nuclear localization sequence of TET3 is shown in the top panel. The subcellular localization of TET3(R1609A), TET3(1–1502), and TET3(1503-C) in HeLa cells was analyzed by immunostaining using anti-FLAG antibody. B, OGT does not affect the nuclear localization of TET3(1503-C) but promotes nuclear export of TET3(680-C). FLAG-TET3(1503-C) and FLAG-TET3(680-C) were expressed alone or together with Myc-OGT, and the subcellular localization of these TET3 proteins was analyzed by immunofluorescence staining using anti-FLAG antibody. C, the failure for OGT to alter TET3(1503-C) nuclear localization is not due to a lack of interaction between OGT and TET3(1503-C). Various FLAG-tagged TET3 constructs were coexpressed with Myc-OGT, and the protein-protein interaction was analyzed by IP-WB as indicated. D, LMB treatment blocks OGT-induced nuclear export of TET3. Myc-OGT and FLAG-TET3 were cotransfected into HeLa cells, and 24 h later the resulting cells were either treated with or without LMB (20 nm), as indicated, for 3 h before the cells were proceeded for immunofluorescence staining.

FIGURE 4.

OGT negatively regulates TET3 5hmC activity, most likely through its effect on TET3 subcellular localization. A, OGT inhibits TET3 enzymatic activity. Myc-TET3 was transfected alone or together with FLAG-OGT, and the ability to catalyze the formation of 5hmC was examined by immunofluorescence staining using an anti-5hmC antibody. Note that, in the cotransfection experiment, the 5hmC staining was substantially weaker for cells with a predominantly cytoplasmic TET3. B, blocking TET3 nuclear export relieves the negative effect of OGT on TET3 activity. HeLa cells were cotransfected with Myc-TET3 and FLAG-OGT for 24 h and then treated without or with 150 μm LMB for 3 h before immunostaining analysis for Myc-TET3 and 5hmC. Note that the cells with strong TET3 cytoplasmic localization (green arrows) were stained much weaker compared with other TET3-positive cells. C, inhibiting the production of cellular UDP-GlcNAc blocks OGT-induced nuclear export of TET3. HeLa cells were cotransfected with Myc-TET3 and FLAG-OGT and treated without or with DON (50 μm) for 8 h. The cells were then proceeded for immunofluorescence staining analysis for TET3 and 5hmC. D, there was no effect of DON treatment on OGT and TET3 expression. HeLa cells were transfected with Myc-TET3 and FLAG-OGT and treated without or with DON (50 μm) for 8 h as in A. The cells were collected, and the expression of Myc-TET3 and FLAG-OGT was determined by WB using anti-Myc and anti-FLAG antibody, respectively.

FIGURE 5.

OGT interacts with and catalyzes the O-GlcNAcylation of TET1 and TET2 but does not affect their subcellular localization and 5hmC enzymatic activity. A and B, OGT interacts with both TET1 and TET2. Myc-TET1C or Myc-TET2 was coexpressed with FLAG-OGT in 293T cells, and the interaction was analyzed by IP-WB. C and D, OGT catalyzes the O-GlcNAc modification of TET1C and TET2. Myc-TET1C and Myc-TET2 were expressed alone or together with FLAG-OGT or FLAG-OGTΔC, as indicated, in 293T cells. The TET proteins were then immunoprecipitated with anti-Myc antibody, and O-GlcNAc modification was detected by WB using anti-O-GlcNAc antibody. E and F, OGT does not affect the nuclear localization and 5hmC activity of full-length TET1 and TET2. Myc-TET1 and Myc-TET2 were expressed alone or together with FLAG-OGT in HeLa cells, and the subcellular localization of TET proteins and 5hmC activity were revealed by immunofluorescence staining.

FIGURE 6.

Glucose metabolism regulates TET3 O-GlcNAcylation and subcellular localization. A, glucose concentration influences TET3 O-GlcNAc modification. HeLa cells were transfected with FLAG-TET3 and cultured with medium containing either 5 mm or 25 mm glucose for 24 h. IP-WB analysis was performed to determine the effect of different glucose concentrations on TET3 O-GlcNAc. B, glucose concentration influences TET3 subcellular localization. HeLa cells were transfected with FLAG-TET3 and cultured with medium containing either 5 mm or 25 mm glucose for 24 h before proceeding to immunofluorescence staining of FLAG-TET3. C, quantitative analysis showing increased TET3 cytoplasmic localization under high-glucose culture conditions. The quantification was made on the basis of the results in B. D, inhibiting OGA activity led to increased global protein O-GlcNAcylation. HeLa cells were treated without or with 150 μm PUGNAc for 24 h and then subjected to WB analysis using anti-O-GlcNAc antibody. E, treatment with PUGNAc led to increased TET3 O-GlcNAcylation. HeLa cells were transfected with or without Myc-TET3 for 24 h and then treated with 150 μm PUGNAc for another 24 h before being processed for IP with anti-Myc antibody, followed by WB using anti-O-GlcNAc antibody. F, treatment with the OGA inhibitor PUGNAc led to increased TET3 cytoplasmic localization. HeLa cells were transfected with Myc-TET3 for 24 h and then treated with or without 150 μm PUGNAc for another 24 h before being processed for immunostaining using anti-Myc antibody. The percentage of cells with a clear cytoplasmic TET3 localization was quantified.