The lab has made significant progress in our understanding of pol IIgamma functions. pol IIgamma does indeed have a novel role in transcription initiation and we are continuing experiments to define this role more precisely. Nevertheless, our hypothesis seems to be correct. Furthermore, we have identified a cycle of GlcNAc addition and removal that is occurring on pol II while it resides on the promoter (hereafter referred to as the G-cycle). Any disruptions of this cycle lead to complete abrogation of transcription. Data thus far indicates this cycle occurs during assembly and recruitment of factors to the promoter. As expected, the pol II CTD is a legitimate substrate for both O-GlcNAc transferase (OGT) and O-GlcNAc aminidase (OGA), the enzymes required for the addition and removal of GlcNAc to and from protein substrates, respectively. We have made considerable progress over the past year and are nearing submission of a manuscript for publication. We have gathered extensive ChIP-seq data showing a remarkable presence of GlcNAc and pol II at promoters but not in the body of genes. We also have detected peaks of the two GlcNAc modification enzymes OGT and OGA, again only on the promoters of genes. These levels vary considerably between promoters. Our most significant observation is that drug inhibitors of OGA (which removes GlcNAc from proteins) causes a complete redistribution of pol II in the genome. Clearly, GlcNAc is required for the proper recruitment of pol II to a promoter. In vitro, we have determined that the serine residues 5 and 7 on the pol II CTD are the sites of GlcNAc modification. This is significant because these two serines are phosphorylated upon the initiation of transcription. Furthermore, drug inhibitors of OGT and OGA, result in the aberrant phosphorylation of the pol II CTD BEFORE transcription initiation. These data suggest that the role of the GlcNAc is in part to prevent premature phosphorylation of pol II CTD. Finally, as one might expect, coimmunoprecipitations show that both OGT and OGA are associated with RNA pol II. The next phase of the project is defined by two questions. Firstly, what are the factors required for pol II/GlcNAc-dependent transcription? We will be approaching this problem using functional biochemistry to dissect the regulation of the GlcNac addition/removal and the factors involved in this process. In vivo, what promoters utilize this GlcNAc-dependent step and does this modification serve as a nutrient sensor, adjusting to the nutrient state of the cell, by directly affecting transcription? Is the GlcNAc regulatory system altered at all in tumor cells or diabetic conditions, where the nutrient state of the cell plays such a direct role in each of these diseases? In any case, our research will pay interesting dividends towards a more complete understanding of promoter regulation in vivo.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Lewis, Brian A; Burlingame, Alma L; Myers, Samuel A (2016) Human RNA Polymerase II Promoter Recruitment in Vitro Is Regulated by O-Linked N-Acetylglucosaminyltransferase (OGT). J Biol Chem 291:14056-61
Lewis, Brian A; Hanover, John A (2014) O-GlcNAc and the epigenetic regulation of gene expression. J Biol Chem 289:34440-8
Ranuncolo, Stella M; Ghosh, Salil; Hanover, John A et al. (2012) Evidence of the involvement of O-GlcNAc-modified human RNA polymerase II CTD in transcription in vitro and in vivo. J Biol Chem 287:23549-61