Moreover, the binding sites of OGT, H2B S112 GlcNAc and TET2 have the highest density around TSS (Fig. TET2 in cells did not alter the TET2-dependent 5hmC synthesis (Supplementary Fig. 3c and Supplementary Fig. 4). Finally, we could not detect glycosylated 5hmC using mass spectrometry (Supplementary Fig. 3d, e and Supplementary Fig. 5). These results suggested that OGT does not affect TET2-dependent 5hmC synthesis. Next, we asked if TET2 regulates the function of OGT. We fractionated ES cell lysates using different salt concentrations and pH levels. A subset of TET2 and OGT could only be eluted from the chromatin by 300 mM NaCl or 0.2 M HCl, indicating that these TET2 and OGT species tightly associate with the chromatin (Supplementary Fig. 6a). Interestingly, knockdown of TET2 by shRNA in ES cells abolished the chromatin-associated OGT, suggesting that TET2 may target OGT to chromatin (Supplementary Fig. 6a). To verify this phenomenon, we used 293T cells stably expressing TET2. Since the exogenous TET2 level was much higher than the endogenous TET2 level (Supplementary Fig. 7), the chromatin-bound OGT was significantly increased in 293T cells stably expressing TET2 (Supplementary Fig. 6b). Moreover, since the D2 mutant of OGT abolished the conversation with TET2, only wild type OGT but not the D2 mutant existed in the chromatin fraction, suggesting that conversation with TET2 is usually important for the chromatin localization of OGT (Supplementary Fig. 6c). In addition, knockdown of OGT PS-1145 by shRNA did not significantly affect the chromatin retention of TET2 (Supplementary Fig. PS-1145 6d). Collectively, these results suggest that TET2 recruits OGT to the chromatin. Recently, it has been shown that histones PS-1145 can be modified by OGT at different sites14-17. Particularly, OGT regulates GlcNAcylation of histone H2B at Serine112 (Fig. 2c). Thus, these data suggest that the conversation between TET2 and OGT is usually important for OGT-dependent histone glycosylation glycosylation assay using wild type OGT and its D2 mutant. With histone octamers as the substrate, both wild type OGT and the D2 mutant only weakly glycosylate histones. The enzymatic activity of wild type OGT was indistinguishable with that of the D2 mutant as both protein can auto-glycosylate themselves (Supplementary Fig. 10). Moreover, supplementation of TET2 did not affect the enzymatic activity of either wild type OGT or the D2 mutant (Fig. 2d). However, when mono-nucleosomes were used as the substrates, supplementation of recombinant TET2 significantly increased the enzymatic activity of wild type OGT but not the D2 mutant (Fig. 2d, Supplementary Fig. 11), suggesting that the conversation with TET2 facilitates OGT to recognize the substrate. Although H3 and H4 were glycosylated glycosylation assay (Supplementary Fig. 11). One possibility is usually that Rabbit Polyclonal to MARK2 glycosylated H2A and H2B suppresses H3 and H4 glycosylation by OGT. Alternatively, the glycosylation sites on H3 and H4 either in the histone octamer or as mono-nucleosomes are not well exposed to the enzyme. In contrast to many other enzymes, OGT only efficiently glycosylates the substrates that it associates with18-21. Thus, it is likely that TET2 recognizes the chromatin and recruits OGT to the chromatin, and the chromatin-associated OGT glycosylates nucleosomal histones at its vicinity. Consistently, only TET2 but not OGT recognizes double-stranded DNA or mono-nucleosome with 5mC (Supplementary Fig. 12). Open in a separate window Physique 2 TET2 enhanced histone glycosylationa, Down-regulation of TET2 impairs H2B GlcNAcylation in ES cells. H2B GlcNAcylation was examined by IP with anti-H2B antibody and Western PS-1145 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 conversation between OGT and TET2 is usually 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 GlcNAcylation assay. All error bars denote s.d., n=3. To examine the distribution of OGT and TET2 around the chromatin of ES cells, we performed genome-wide ChIP sequencing analysis (ChIP-seq) using anti-OGT, anti-H2B S112 GlcNAc and anti-mTET2 antibodies. We validated our ChIP-seq results using ChIP-qPCR to examine 45 different loci that represent a broad range of ChIP-seq fragment counts (Supplementary Fig. 13). Next, we compared the TET2 target genes with a published hmeDIP database3, and found that 47 % of hmC.
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