Evidence of the involvement of O-GlcNAc-modified human RNA polymerase II CTD in transcription in vitro and in vivo

J Biol Chem. 2012 Jul 6;287(28):23549-61. doi: 10.1074/jbc.M111.330910. Epub 2012 May 17.

Abstract

The RNA polymerase II C-terminal domain (CTD), which serves as a scaffold to recruit machinery involved in transcription, is modified post-translationally. Although the O-GlcNAc modification of RNA polymerase II CTD was documented in 1993, its functional significance remained obscure. We show that O-GlcNAc transferase (OGT) modified CTD serine residues 5 and 7. Drug inhibition of OGT and OGA (N-acetylglucosaminidase) blocked transcription during preinitiation complex assembly. Polymerase II and OGT co-immunoprecipitated, and OGT is a component of the preinitiation complex. OGT shRNA experiments showed that reduction of OGT causes a reduction in transcription and RNA polymerase II occupancy at several B-cell promoters. These data suggest that the cycling of O-GlcNAc on and off of polymerase II occurs during assembly of the preinitiation complex. Our results define unexpected roles for both the CTD and O-GlcNAc in the regulation of transcription initiation in higher eukaryotes.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine / analogs & derivatives
  • Acetylglucosamine / metabolism*
  • Acetylglucosamine / pharmacology
  • Acetylglucosaminidase / antagonists & inhibitors
  • Acetylglucosaminidase / metabolism
  • Binding Sites
  • Blotting, Western
  • Cell Line, Tumor
  • Chromatin Immunoprecipitation
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation / drug effects
  • HeLa Cells
  • Humans
  • N-Acetylglucosaminyltransferases / antagonists & inhibitors
  • N-Acetylglucosaminyltransferases / genetics
  • N-Acetylglucosaminyltransferases / metabolism
  • Oximes / pharmacology
  • Phenylcarbamates / pharmacology
  • Promoter Regions, Genetic / genetics
  • Protein Binding
  • Protein Processing, Post-Translational*
  • Protein Subunits / metabolism
  • RNA Interference
  • RNA Polymerase II / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Serine / metabolism
  • Transcription, Genetic / drug effects
  • Transcription, Genetic / genetics*

Substances

  • Enzyme Inhibitors
  • Oximes
  • Phenylcarbamates
  • Protein Subunits
  • N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime
  • Serine
  • N-Acetylglucosaminyltransferases
  • O-GlcNAc transferase
  • RNA Polymerase II
  • Acetylglucosaminidase
  • Acetylglucosamine