Initiation And Elongation Of RNA Polymerase II Are Regulated By Multiple OGlcNAcylation Events

RNA pol II is thought to be regulated at two positions on a gene and this regulation determines whether a gene is transcribed. The first is at the promoter, where pol II and the general transcriptional machinery are recruited to form a pre‐initiation complex (PIC). Upon receiving the appropriate signal, pol II initiates transcription and elongates into the gene body. The current dogma states that a hypophosphorylated RNA pol II (pol IIA) is the initiation‐competent species. However, this pol IIA was shown to also contain glcnacylated pol II. Thus, although the O‐GlcNAc modification of the RNA polymerase II CTD was described twenty years ago it has never been clear what the initiation‐specific species of RNA pol II is, or whether O‐GlcNAcylation regulates PIC formation. The second position is located immediately downstream of the transcriptional start site (TSS). In the late 1980s, a transcriptionally engaged, paused pol II was found on several genes, roughly at +50 relative to the TSS. Recent genome‐wide approaches have shown that paused pol II exists on approximately 40% of promoters in the genome and thus understanding the regulation of pausing is a major issue in eukaryotic transcription. It is not known nor has it been suspected that O‐GlcNAcylation plays a role in regulating these events. We have made significant advances in showing functional requirements for O‐GlcNAcylation during both transcription initiation and elongation. Firstly, we have collected several lines of evidence that O‐GlcNAcylation events are required for transcription initiation and the recruitment of RNA pol II to promoters. Notably, OGT and OGA chemical inhibitors block transcription in vitro and inhibition of O‐GlcNAcylation prevents pol II entry into the promoter in vitro and in vivo. Secondly, we have found that chemical inhibition of the O‐GlcNAc aminidase (OGA) has pausing and elongation defects in vivo. In vitro elongation assays also showed a functional requirement for OGA activity in both crude nuclear extract and partially purified systems. Lastly, we identified a 600 kDa complex containing OGA and the pausing/elongation factor DSIF. These data indicate that O‐GlcNAcylation is essential for RNA pol II promoter recruitment and that RNA pol II goes through a cycling of O‐GlcNAcylation at the promoter, where before initiation pol II is in a glcnacylated state, and upon initiation, the O‐GlcNAc is removed. Our data also suggest a novel role of dynamic cycling of O‐GlcNAcylation in RNA pol II elongation control by showing a functional and physical coupling of OGA with the pol II elongation and the pausing machinery. In conclusion, our work has brought to light an entirely new level of regulation of both transcription initiation and elongation, mediated by O‐GlcNAcylation enzymes. These results are possibly manifestations of a direct link between the regulation of RNA pol II activity genome‐wide and the nutrient state of the cell. Support or Funding Information CCR/NCI/NIH Intramural Research Program