Elucidating the signaling and protein interaction networks of the O-GlcNAc transferase during embryonic stem cell state transitions - Project Summary/Abstract O-GlcNAc is a single N-acetylglucosamine coupled to serine and threonine residues of nuclear and cytoplasmic proteins. Analogous to phosphorylation, O-GlcNAc signaling is dynamic, rapidly added and removed from proteins in a site-specific manner in response to cellular perturbations and extracellular cues. Because both modifications occur on the same residues it is hypothesized that there is a functional crosstalk between O- GlcNAc and phosphorylation, where one may affect deposition or removal the other. Unlike phosphorylation, however, which is catalyzed by over 500 kinases and roughly 300 phosphatases, the mammalian genome only encodes a single O-GlcNAc transferase (OGT) and a single hydrolase (OGA). While many kinases recognize specific amino acid sequences in their substrates, the determinants guiding OGT are unclear and likely manifold. This intracellular glycosylation is implicated in nearly every cellular process from gene expression and signal transduction to cell division and differentiation. Despite the ubiquitous nature of this post-translational modification in health and disease, the specific functions of OGT and the basic principles of O-GlcNAc signaling remain almost entirely elusive. This gap in our knowledge has been largely due to the major lack of tools and technologies available to study O-GlcNAc signaling or perturb the essential OGT. Here, we aim to uncover the basic principles of OGT and O-GlcNAc signaling, and their role in transcriptional regulation of cellular differentiation. We have recently developed a highly sensitive and specific enrichment reagent to analyze O-GlcNAc-modified peptides from cells and tissues by mass spectrometry. Using these new anti-O-GlcNAc antibodies we will elucidate the global, site-specific temporal dynamics of O-GlcNAc signaling during the transition from totipotency to naïve and primed pluripotency. Combined with phosphoprotemic profiling of the same samples, we will monitor for crosstalk between these two post-translational modifications. To gain insight into how OGT targets its diverse array of substrates, we will deconvolute the extensive OGT interactome employing chemical crosslinking and biochemical fractionation, followed by mass spectrometric analysis. To explore how OGT uses adaptor proteins to targets substrates, we will use an innovative approach, degrading specific OGT interacting proteins and assessing changes in downstream O-GlcNAc signaling using our new quantitative glycoproteomic approach. Integrating these two research programs, we will create a holistic, high- resolution understanding of the principles of O-GlcNAc signaling. The MIRA mechanism will not only enable the investigation of basic O-GlcNAc biology, but will provide the flexibility to conduct data-driven follow-up, functional analyses of dynamic O-GlcNAc/crosstalk sites and distinct OGT complexes, and their role in transcriptional regulation of some of the earliest development decisions.
