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 O-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 have published our first paper on this topic. In vitro, we have shown that inhibitors of the addition and removal of O-GlcNAc disrupt transcription, specifically the formation of PICs at the promoter. We have shown that pol II is a substrate for OGT and OGA. We have determined that the serine residues 5 and 7 on the pol II CTD are the sites of O-GlcNAc modification. This is significant because these two serines are phosphorylated upon the initiation of transcription. This suggests that the conversion of the CTD from a glcnacylated CTD to a CTD ready for initiation of transcription (and hence phosphorylation) is a regulated step during transcription. Finally, as one might expect, coimmunoprecipitations show that both OGT and OGA are associated with RNA pol II and several other proteins necessary for the initiation of transcription. Our most significant observation is that shRNA-mediated reduction in OGT causes a decrease in transcription and in the occupancy of RNA pol II at the promoter. Clearly, O-GlcNAc is required for the proper recruitment of pol II to a promoter. We have also gathered extensive ChIP-seq data showing a remarkable presence of O-GlcNAc and pol II at promoters but not in the body of genes. We also have detected peaks of the two O-GlcNAc modification enzymes OGT and OGA, again only on the promoters of genes. These levels vary considerably between promoters. We now have data that glcnacylated pol II can be detected on promoters in vivo, a "smoking gun" if you will, for the existence of this pol II species in vivo. Our ChIP-seq analysis also shows that B-cell specific genes are not overrepresented in genes that are glcnacylated. This suggests that there are important regulatory differences between genes that are glcnacylated and those that are not. Finally, we have shown that the levels of glcnacylated pol II decrease in cells starved of glucose and which can then increase with the addition of glucose or glucosamine. Glucose is used to make UDP-GlcNAc and glucosamine is a direct intermediate in the synthesis of UDP-GlcNAc (the substrate for OGT). These results suggest a direct link between the nutrient state of the cell and glcnacylated pol II and the levels of pol II on promoters. This in turn shows that the ramifications of hyperglycemia are potentially manifested across the genome, affecting thousands of genes and the pol II density on them. Then end result then are changes in the amount of mRNA synthesized in the genome. As such this genome-wide dysregulation may have far-reaching consequences in type II diabetes, characterized by hyperglycemia. We have now completed our ChIP-seq analysis showing that their is a very high concentration of O-GlcNAc on approximately 40% of promoters in the human genome and its position on promoters overlaps the position of promoter-localized RNA pol II. More imporantly, we have shown that O-GlcNAcylated pol II is a promoter-localized species both in transcriptionally active nuclear extracts and also in vivo. These data are the first to define a new species of pol II on promoters and overturn the current dogma that no such species exists. Lastly, we now have shown that the nutrient state of the cell is reflected in the amount of O-GlcNAcylation of pol II and that this in turn from OGT and OGA knockdowns controls the amount of pol II at promoters. Thus O-GlcNAcylation of pol II serves as a nutrient sensor that is localized to promoters across the genome. This means that pol II levels are responding to the nutrient state of the cell, much like a rheostat. Lastly, we have also shown that the metabolic organs, liver and muscle, display unique fluctuations in O-GlcNAcylation of polII and that this flux is regulated by insulin. This goes towards establishing a direct link between pol II O-GlcNAcylation and genome-wide disruptions in transcription in diabetes. We are continuing these studies now. In addition we have achieved much in understanding the regulation of this behavior by the O-GlcNAc removal enzyme, OGA. We expect both of these narratives to be published in the upcoming fiscal year. The next phase of the project is defined by two questions. Firstly, what are the factors required for pol II/O-GlcNAc-dependent transcription? We will be approaching this problem using functional biochemistry to dissect the regulation of the O-GlcNac addition/removal and the factors involved in this process. In vivo, what promoters utilize this O-GlcNAc-dependent step and to what extent does the O-GlcNAc modification serve as a nutrient sensor, adjusting to the nutrient state of the cell, by directly affecting transcription? Is the O-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. This project is an interesting example of the convergence of basic research on physiology. We began by trying to understand the nature, function, and the regulation of this novel species of pol II characterized by the O-GlcNAc modification of its C-terminal domain. It is becoming clear that this species plays an important role in the beginning of transcription of genes. But it is now clear that the link between O-GlcNAc and pol II is more than that and is likely massively perturbed in the cells and genomes of those with insulin-resistance and adult onset type II diabetes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011223-05
Application #
8763412
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2013
Total Cost
$835,518
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code