A novel form of the human RNA polymerase II (pol II) was described 25 years ago and yet its function remains unknown. This form is defined by O-GlcNAc modification of the pol II C-terminal domain (CTD). O-GlcNAcylation is a common but largely ignored modification of proteins. There are two enzymes involved here: the O-GlcNAc transferase (OGT) adds GlcNAc to a protein substrate while the O-GlcNAc aminidase removes the GlcNAc. The O-GlcNAc modification of pol II (pol IIgamma) poses an obvious contradiction to the dogma that the unmodified form of pol II, pol IIA, is the initiation-specific form of the enzyme. Our data indicate that the pol IIgamma is involved in the initiation of transcription either solely or in conjunction with pol IIA. Additionally, we have shown convincingly that OGT and OGA activities are required for initiation of transcription. More recent work over the past fiscal year has expanded our understanding, and we can now show direct effects of OGT and OGA on the regulation of transcription elongation. This is incredibly important finding in that at least 50% of genes in the genome are regulated by modulating elongation of RNA pol II through the gene body. Additionally, we have identified a cycle of O-GlcNAc addition and removal that is occurring on promoters (hereafter referred to as the G-cycle). Disruption of this cycle leads to the complete abrogation of transcription. The G-cycle connects to an even more important finding: we have shown that the levels of glcnacylated pol II and pol II promoter occupancy 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 pol II. This in turn shows that the ramifications of hyperglycemia in type II diabetes are potentially manifested across the genome, affecting thousands of genes and the pol II density on them. The end result is changes in the amount of mRNA synthesized in the genome. Thus O-GlcNAcylation of pol II may serve 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 thermostat is used to change the temperature in a room. Nutrient states and their regulation also apply to cancer cells, whose metabolism is drastically altered. We would expect glcnacylation levels and regulation to be altered as well and these connections offer further avenues of exploration into the regulation of a cancer phenotype by glcnacylation. Indeed, cellular glcnacylation is severely altered in several malignant cell types. Lastly, we have achieved much in understanding the regulation of the O-GlcNAc removal enzyme, OGA. Over the past 12 months, we have found that chemical inhibition of OGA affects the elongation of two sets of genes. The most important class of genes in this regard is genes with paused polymerases. These paused polymerases are transcriptionally engaged about 50 base pairs downstream of the transcriptional start site. This is an important regulatory feature of many proto-oncogenes such as c-myc, fos, and jun. We found that these polymerases are aberrantly released upon OGA inhibition and without the proper signal to do so. Thus OGA is a necessary component of the regulation of paused genes which account for more than half of genes in the genome. Our in vitro transcription biochemistry confirms our in vivo results and also showed that OGA is in a complex with a factor necessary for pol II pausing. This has been an exciting and completely unexpected set of discoveries over the past year and which is of broad interest in the gene regulation field. In summary, we have made several important discoveries. The first the regulation of transcription in initiation by O-GlcNAcylation, and it being specifically required for pol II recruitment to promoters. The second is a completely unexpected regulatory connection between glcnacylation and RNA pol II elongation. The third is the existence of a dynamic cycle of glcnacylation at promoters that connects with our fourth discovery that RNA pol II, via O-GlcNAcylation is acting a sensor of the nutrient state of the cell across the genome. As far as basic research goes, the work has been an important success. It has challenged an existing paradigm and created a new model of how genes are regulated. Furthermore, it has made novel discoveries and connections that were not predicted or expected to exist. The most recent work then has created an entirely new paradigm for understanding the regulation of RNA pol II during its elongation phase through a gene. These discoveries for the first time show a direct connection between O-GlcNAcylation and the regulation of proto-oncogene expression and emphasize the importance of basic scientific research in establishing unexpected connections between disparate biological phenomena. Such research can only help in offering insights for more translational and clinical studies.
NIH ExPorter
NIH ExPorter