Human RNA Polymerase II Promoter Recruitment in Vitro Is Regulated by O-Linked N-Acetylglucosaminyltransferase (OGT)

Brian A Lewis,Alma L Burlingame,Samuel A Myers

doi:10.1074/jbc.m115.684365

Abstract

Although the O-linked N-acetylglucosamine (O-GlcNAc) modification of the RNA polymerase II C-terminal domain was described 20 years ago, the function of this RNA polymerase II (pol II) species is not known. We show here that an O-GlcNAcylated pol II species (pol IIγ) exists on promoters in vitro Inhibition of O-GlcNAc-transferase activity and O-GlcNAcylation prevents pol II entry into the promoter, and O-GlcNAc removal from pol II is an ATP-dependent step during initiation. These data indicate that O-GlcNAc-transferase activity is essential for RNA pol II promoter recruitment and that pol II goes through a cycling of O-GlcNAcylation at the promoter. Mass spectrometry shows that serine residues 2 and 5 of the pol II C-terminal domain are O-GlcNAcylated, suggesting an overlap with the transcription factor IIH (TFIIH)-dependent serine 5 phosphorylation events during initiation and P-TEFb (positive transcriptional elongation factor b) events during elongation. These data provide unexpected and important insights into the role of a previously ill-defined species of RNA polymerase II in regulating transcription.

Highlights

We found the first evidence that serine 2 residues can be O-GlcNAcylated
ConclusionThe existence of pol II␥ on promoters and the transcriptional requirement for O-GlcNAc and its cycling are both unexpected and outside the paradigms of transcriptional regulation

Summary

Methods

In Vitro PIC Assays—PICs were formed by adding nuclear extract, BC buffer (20 mM Tris, pH 7.9 at 4 °C, 10% glycerol, 0.2 mM EDTA, 0.2 mM DTT, 100 mM KCl) containing 100 mM KCl (BC100), 2 ␮g of Escherichia coli DNA to E3 promoter DNA immobilized on M280 Dynabeads that were blocked for 30 min using 100 mg/ml BSA in BC100 [5]. PICs were incubated as indicated with 0.1 mM STO45849 or DMSO (0.5 ␮l of 10 mM STO45849 stock in DMSO or 0.5 ␮l of DMSO control) or 3 mM PUGNAc. For the wheat germ agglutinin affinity purification assays, assembled PICs on the adenovirus E3 promoter were washed in BC100, 0.05% Nonidet P-40 and eluted with 100 ␮l of BC buffer containing 500 mM KCl (BC500). For the wheat germ agglutinin affinity purification assays, assembled PICs on the adenovirus E3 promoter were washed in BC100, 0.05% Nonidet P-40 and eluted with 100 ␮l of BC buffer containing 500 mM KCl (BC500) This was diluted 2ϫ with H2O and incubated for 1 h at room temperature with the equivalent of 20 ␮l of WGA-agarose slurry (Vector Laboratories) or for 1 h with 1 ␮l of 8WG16 mAb (Covance) and protein G beads (Roche Applied Science). To detect O-GlcNAcylated pol II using 110.6 Western blotting (Fig. 1D, right panel), the PIC assay used 400 ng of DNA template

Results

Discussion

Conclusion