RNA polymerase II O-GlcNAcylation promotes nuclear entry to drive transcription

Abstract

O-GlcNAcylation of RNA Polymerase II (RNA Pol II) was described in 1993 and here we wanted to probe the functional significance of this modification. O-GlcNAc transferase (OGT) is the sole enzyme performing nucleo-cytoplasmic O-GlcNAcylation. The role of RNA Pol II O-GlcNAcylation has remained enigmatic partly due to the lack of reagents selectively recognizing the O-GlcNAcylated polymerase. We developed a novel approach, wheat germ agglutinin (WGA) lectin-based enrichment for O-GlcNAcylated proteins followed by traditional ChIP for RNA Pol II, to identify chromatin-binding sites of the O-GlcNAcylated RNA Pol II. We termed this technique as wChIP and used it to demonstrate that the O-GlcNAcylated RNA Pol II is enriched to the promoters of highly expressed genes. RNA Pol II has a repetitive c-terminal domain (CTD) of YSPTSPS, which is heavily post-translationally modified. Using structural modeling and WGA pulldown-experiments, we identify Ser-5 as a CTD site that is dynamically modified by OGT. Mass spectrometry-based detection of proteins that recognize CTD that is O-GlcNAcylated on Ser-5 identified nuclear transport protein, karyopherin subunit beta 1 (KPNB1), as a reader protein for this modification. Functionally, high activity of both OGT and KPNB1 is required for the efficient entry of RNA Pol II to nucleus and chromatin. OGT and KPNB1 are significantly co-expressed in prostate cancer tissue, and their combined inhibition is more toxic to prostate cancer cells than normal prostate cells. We propose that RNA Pol II O-GlcNAcylation promotes entry to nucleus, and this could integrate metabolic information to regulate global transcription.