Evidence of the Involvement of O-GlcNAc-modified Human RNA Polymerase II CTD in Transcription in Vitro and in Vivo

Metabolism Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2012; 287(28):23549-61. DOI: 10.1074/jbc.M111.330910
Source: PubMed


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.

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    • "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. "
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    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
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    • "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. "
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    ABSTRACT: Eukaryote's RNA polymerases II (RNAPII) have the feature to contain, at the carbossi-terminal region of their largest subunit Rpb1, a unique CTD domain. Rpb1-CTD is composed of an increasing number of repetitions of the Y1 S2 P3 T4 S5 P6 S7 heptad that goes in parallel with the developmental level of organisms. Because of its composition, the CTD domain has a huge structural plasticity; virtually all the residues can be subjected to post-translational modifications and the two prolines can either be in cis or trans conformations. In light of these features, it is reasonable to think that different specific nuances of CTD modification and interacting factors take place not only on different gene promoters but also during different stages of the transcription cycle and reasonably might have a role even if the polymerase is on or off the DNA template. Rpb1-CTD domain is involved not only in regulating transcriptional rates, but also in all co-transcriptional processes, such as pre-mRNA processing, splicing, cleavage and export. Moreover, recent studies highlight a role of CTD in DNA replication and in maintenance of genomic stability and specific CTD-modifications have been related to different CTD functions. In this paper we examine results from the most recent CTD-related literature and give an overview of the general function of Rpb1-CTD in transcription, transcription-related and non transcription-related processes in which it has been recently shown to be involved in. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 05/2014; 229(5). DOI:10.1002/jcp.24483 · 3.84 Impact Factor
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    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.
    RNA biology 09/2012; 9(9). DOI:10.4161/rna.21726 · 4.97 Impact Factor
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