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.
"By definition, this structure also excludes the elongating form of RNA polymerase II (Phospho-S), although we show non-elongating RNA polymerase II in this region by pan RNA polymerase II antibody staining. As previously demonstrated, RNA polymerase II is O-GlcNAcylated on the C-terminal domain and O-GlcNAcylated RNA polymerase II is a transient state during the initiation complex formation (Ranuncolo et al., 2012). O-GlcNAcylation of RNA polymerase II is thought to occur in pre-initiation state before CTD kinases may act. "
[Show abstract][Hide abstract] ABSTRACT: O-GlcNAc Transferase (OGT) catalyzes protein O-GlcNAcylation, an abundant and dynamic nuclear and cytosolic modification linked to epigenetic regulation of gene expression. The steady-state levels of O-GlcNAc are influenced by extracellular glucose concentrations suggesting that O-GlcNAcylation may serve as a metabolic sensor. Intriguingly, human OGT is located on the X-chromosome (Xq13) close to the X-inactivation center (XIC), suggesting that OGT levels may be controlled by dosage compensation. In human female cells, dosage compensation is accomplished by X-inactivation. Long noncoding RNAs and polycomb repression act together to produce an inactive X chromosome, or Barr body. Given that OGT has an established role in polycomb repression, it is uniquely poised to auto-regulate its own expression through X-inactivation. In this study, we examined OGT expression in male, female and triple-X female human fibroblasts, which differ in the number of inactive X chromosomes (Xi). We demonstrate that OGT is subjected to random X-inactivation in normal female and triple X cells to regulate OGT RNA levels. In addition, we used chromatin isolation by RNA purification (ChIRP) and immunolocalization to examine O-GlcNAc levels in the Xi/Barr body. Despite the established role of O-GlcNAc in polycomb repression, OGT and target proteins bearing O-GlcNAc are largely depleted from the highly condensed Barr body. Thus, while O-GlcNAc is abundantly present elsewhere in the nucleus, its absence from the Barr body suggests that the transcriptional quiescence of the Xi does not require OGT or O-GlcNAc.
Frontiers in Genetics 08/2014; 5:256. DOI:10.3389/fgene.2014.00256
"Only recently, at the formation of transcription preinitiation complex, glycosylated CTD on serine residues (Ser5/ Ser2-G) has been demonstrated. Since glycosylation and phosphorylation are mutually exclusive, these data suggest a functional role of Ser2/Ser5-G in preventing phosphorylation of CTD before its recruitment on promoters (Ranuncolo et al., 2012). As already mentioned, residues at position 7 of nonconsensus heptads are mainly arginines or lysines. "
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic RNA polymerase II (RNAP II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation of Ser2, Ser5 and Ser7 residues orchestrates the binding of transcription and RNA processing factors to the transcription machinery. Recent studies show that the two remaining potential phosphorylation sites, tyrosine-1 and threonine-4, are phosphorylated as well and contribute to the previously proposed "CTD code". With the impairment of binding of CTD interacting factors, these novel phosphorylation marks add an accessory layer of regulation to the RNAP II transcription cycle.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.