The cycling of N-acetylglucosamine on Ser(Thr) residues (O-GlcNAcylation) of nuclear, cytoplasmic and mitochondrial proteins serves as a nutrient sensor to regulate signaling, transcription, and cellular physiology. Abnormal O-GlcNAcylation underlies the etiology of diabetes, cancer and Alzheimer's disease. O-GlcNAcylation regulates nearly every aspect of transcription, including RNA polymerase II, histones, DNA methyltransferases, and nearly all transcription factors. Yet we know very little about the mechanisms involved. TATA-binding protein (TBP) is amongst the most important components of the transcription machinery. Our recent studies indicate that TBP regulated by O-GlcNAcylation in response to nutrients. O-GlcNAcylation has extensive crosstalk with protein phosphorylation and other modifications. However, unlike phosphorylation, which is catalyzed by hundreds of kinases, there is only one highly conserved gene encoding O-GlcNAc transferase (OGT). Nonetheless, OGT site-specifically modifies thousands of proteins. Our data indicate that OGT's substrate specificity is not only determined by its specificity for peptide sequence, but also by its transient associations with other proteins, which dynamically target it to specific substrates. OGT targeting interactions are largely mediated by its tetratricopeptide repeats (TPRs). OGT missing TPRs remains active against small peptides, but has poor activity against full-length proteins. Herein we will investigate two major questions related to O-GlcNAcylation: Specific Aim 1: Continue to Study Glucose Regulation of TATA-binding protein (TBP) via its O-GlcNAcylation. Hypothesis: At certain promoters, glucose regulates TBP DNA-binding and its ability to bend DNA by altering its O- GlcNAcylation. We will systematically elucidate mechanisms and functions of this nutrient regulation of TBP. Three goals: A. Roles of O-GlcNAcylation in the molecular and cellular properties of TBP? B. Roles of O- GlcNAc in TBP's Interactions in the Transcription Cycle. C. O-GlcNAc's In Vivo Roles in TBP-mediated Gene Expression. Specific Aim 2: Continue to Elucidate How OGT is Specifically Targeted to Thousands of Different Protein Substrates? Hypothesis: OGT achieves a high degree of specificity not only by its recognition of peptide sequence, but also by it being targeted to substrates by accessory proteins. This Aim will systematically evaluate both the general and specific roles of OGT binding partners in OGT's activities toward its many substrates. Given O-GlcNAc's importance to mechanisms of chronic disease, such as glucose toxicity in diabetes, cancer and neurodegeneration, elucidation of these mechanisms will not only be key to understanding transcription and signaling, also to uncovering new avenues for therapeutics.