The cycling of N-acetylglucosamine on Ser(Thr) residues (O-GlcNAcylation; OGN) on nuclear, cytoplasmic and mitochondrial proteins serves as a nutrient sensor to regulate signaling, transcription, and cellular physiology. Abnormal OGN underlies the etiology of diabetes, cancer and Alzheimer's disease. OGN regulates nearly every aspect of transcription in response to nutrients. Great strides have been made in developing methods that elucidate the functions of OGN. While we can increase or decrease global OGN in cells, the greatest impediment toward a mechanistic understanding of OGN's functions is the lack of a method to alter OGN on a single protein without affecting the other thousands of OGN proteins within a cell. We discovered that the C-terminal domain of RNA polymerase II, which consists of 52 imperfect repeats of the sequence, YSPTSPS, is heavily OGN when it is not phosphorylated. OGN of the CTD is required for transcription initiation, is reciprocal with phosphorylation, and the sugar must be removed by O-GlcNAcase prior to elongation. While there have been many studies of the role of phosphorylation of the CTD in transcription, in contrast, there have been no studies of the specific roles of OGN on the CTD! We propose to develop an optogenetic approach to specifically target the O-GlcNAc transferase (OGT) to specific proteins. Our plan is to adapt the LOV2 light-inducible dimer (iLID) system. In this system, light-induced molecular association occurs on the sub-second time scale and reversion in the dark can occur within ten minutes. Initially, we will use iLID to investigate the roles of OGN in the insulin signaling pathway. OGT is normally targeted to its substrates by accessory proteins, among which are TET proteins, enzymes that hydroxymethylate cytosine residues, but also target OGT to chromatin. We will further investigate the roles of TET proteins in OGT actions on chromatin, particularly on the CTD of Pol II. Finally, we will study the roles of OGN on the CTD of RNA pol II in terms of its nutrient and stress responsiveness, and cell type differences in sites modified. We will elucidate the interactome of OGN-CTD and we will determine if OGN plays a role in RNA pol II pausing at promoters. These studies are not only elucidating molecular mechanisms of how nutrients regulate transcription, but they also are key to revealing how hyperglycemia, as occurs in diabetes, abnormally alters gene expression in many tissues. Molecular mechanisms revealed in these studies will likely lead to totally novel targets for the treatment of chronic diseases of aging, particularly diabetes.
Decades of work have shown that a cycling sugar modification of proteins in the nucleus and cytoplasm of cells is a major mechanism by which what we eat regulates our cellular physiology. In fact, dysregulation of this sugar cycling contributes to major diseases of aging, such as diabetes, cancer and Alzheimer?s disease. This project will develop novel tools to elucidate the functions of this modification, and it will reveal mechanisms of how nutrients regulate gene expression.
