TET2 promotes histone O-GlcNAcylation during gene transcription

Abstract

Ten eleven translocation (TET) enzymes, including TET1, TET2 and TET3, convert 5-methylcytosine to 5-hydroxymethylcytosine and regulate gene transcription. However, the molecular mechanism by which TET family enzymes regulate gene transcription remains elusive. Using protein affinity purification, here we search for functional partners of TET proteins, and find that TET2 and TET3 associate with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), an enzyme that by itself catalyses the addition of O-GlcNAc onto serine and threonine residues (O-GlcNAcylation) in vivo. TET2 directly interacts with OGT, which is important for the chromatin association of OGT in vivo. Although this specific interaction does not regulate the enzymatic activity of TET2, it facilitates OGT-dependent histone O-GlcNAcylation. Moreover, OGT associates with TET2 at transcription start sites. Downregulation of TET2 reduces the amount of histone 2B Ser 112 GlcNAc marks in vivo, which are associated with gene transcription regulation. Taken together, these results reveal a TET2-dependent O-GlcNAcylation of chromatin. The double epigenetic modifications on both DNA and histones by TET2 and OGT coordinate together for the regulation of gene transcription.

Figure 1. TET2 forms a complex with OGT

a, Purification of TET2 and TET3-associated proteins. TET2 and TET3-associated proteins were analyzed by mass spectrometry and are shown in the Supplementary Fig. 1a. b, OGT interacts with TET2 and TET3 but not TET1. SBP tagged TET1-3 were expressed and examined with indicated antibodies. The whole cell lysates (WCL) was used as the input. c, OGT interacts with TET2 endogenously in ES cells. Irrelevant IgG was used as the IP controls (Con). PALB2 was used as the negative control. d, The DSBH domain of TET2 interacts with OGT. Deletion mutants of TET2 mutants were expressed. The F3 mutant with the DSBH domain (catalytic domain) interacts with OGT. e, TPR5 and 6 of OGT interacts with TET2. The D2 mutant of OGT that is deleted TPR5 and 6 abolished the interaction with TET2. f, OGT directly binds TET2. Sf9 cells were infected with baculoviruses encoding SBP-OGT and/or GST-TET2 catalytic domain (TET2CD). The protein complex was purified by streptavidin beads or GST beads and examined by coomassie blue staining.

Figure 2. TET2 enhanced histone glycosylation

a, Down-regulation of TET2 impairs H2B GlcNAcylation in ES cells. H2B GlcNAcylation was examined by IP with anti-H2B antibody and Western blot with anti-GlcNAc antibody (RL2) or anti-H2B S112 GlcNAc antibody. Histogram shows the relative level of H2B S112 GlcNAc in TET2 down-regulated cells compared to that in control shRNA treated cells. b, Up-regulation of wild type TET2 or TET2 enzymatic dead mutant (H1382Y/D1384A) induced H2B S112 GlcNAcylation in 293 cells. c, The interaction between OGT and TET2 is important for H2B GlcNAcylation and H2B S112G GlcNAcylation. Wild type OGT, the enzymatic dead mutant of OGT (G482S) and the D2 mutant were expressed in 293T cells. H2B GlcNAcylation and H2B S112 GlcNAcylation were examined. d, TET2 facilitated OGT-dependent histone glycosylation in mono-nucleosome but not in recombinant core histones. Tritium-labeled GlcNAc was incorporated into the histones in the in vitro GlcNAcylation assay. All error bars denote s.d., n=3.

Figure 3. TET2 regulates H2B S112 GlcNAc and gene transcription

a, Venn diagram shows a significant overlap between OGT, H2B S112 GlcNAc and TET2 target genes. b, Mean distribution of tags at gene TSS (± 4 kb). c, Examples of OGT, H2B S112 GlcNAc and TET2 ChIP-seq results in mouse ES cells. d, ChIP-qPCR was performed to examine TET2, OGT and H2B S112 GlcNAc in control, TET2 knockdown or OGT knockdown cells (shCon, shTET2 and shOGT). e, The expression of TET2 and OGT common target genes is higher than average gene expression in ES cells. Data analysis was explained in the Material and Method. Boxplots show median, 25^th and 75^th percentile expression levels in ES cells. p<0.00001. f, Loss of TET2 or OGT is associated with the reduced expression of their common target genes. All error bars denote s.d., n=3.