Linking Maternal Sugar Consumption to Progenies’ Developmental Defects: A Focus on OTX2’s O‐GlcNAcylation

Abstract

The O‐GlcNAc post‐translational modification is a dynamic and nutrient‐sensing modification that is reversibly added onto nuclear, cytoplasmic, and mitochondrial proteins. O‐GlcNAc is a unique glucose rheostat for cell signaling, since the level of extracellular glucose is reflected by the level of UDP‐GlcNAc, the primary substrate for O‐GlcNAcylation. To date, thousands of O‐GlcNAcylated proteins have been identified. Numerous physiological processes are O‐GlcNAc‐regulated such as cell cycle, transcriptional/translational regulation, protein localization and degradation. Moreover, O‐GlcNAc deregulation has been linked to pathologies like diabetes, cardiovascular diseases, neurodegeneration and cancers. O‐GlcNAcylated proteins are often involved in major signaling pathways, including those involved in development. Studying this modification therefore allows us to investigate the impact that glucose levels in the diet have in development, by regulating protein function in response to glucose concentration.Using cellular and mouse models, our lab has previously delved into the consequences of hyper‐O‐GlcNAcylation in the brain. Among phenotypes like early onset obesity and growth defects, the pituitary gland of these mice was generally smaller and corresponded with a developmental delay. In this study, we investigate the specific effects of hyper‐O‐GlcNAcylation in the pituitary gland by knocking out the O‐GlcNAcase (Oga), the enzyme that removes the O‐GlcNAc modification. We found that pituitary specific Oga KO mice had impaired eye development, growth defects, and inability to be metabolically self‐sufficient. A critical aspect of pituitary’s ontogeny is the local transient expression of the homeobox protein OTX2, which was identified as a major O‐GlcNAcylated protein in the developing brain, and interestingly, was dysregulated in our brain hyper‐O‐GlcNAcylated models. Using specific domain constructs, we have discovered that the Repression Domain of OTX2 is highly O‐GlcNAcylated, potentially affecting transcriptional activity and critical phosphorylation sites of this protein. To assess differential OTX2’s phosphorylation upon hyper‐O‐GlcNAcylation in vitro, we treated HeLa cells with the OGA inhibitor Thiamet‐G. After immunoprecipitation, OTX2’s phosphorylation was assessed using a Phos‐tag gel, delaying specifically the migration of the phosphorylated protein. Surprisingly, we did not observe a significant difference in OTX2 phosphorylation with O‐GlcNAc fluctuation. This suggested that OTX2’s O‐GlcNAcylation does not compete with phosphorylation sites and rather regulates OTX2’s activity or localization.To summarize, this study highlights the O‐GlcNAc modification as a nutrient‐dependent sensor that regulates the homeobox protein OTX2 during pituitary and brain development. Like many homeobox proteins, OTX2 level needs to be tightly regulated for proper patterning and development, and its deregulation amongst other proteins is a major driver of Medulloblastoma. Therefore, we also foresee that O‐GlcNAcylated OTX2 may play a major role in Medulloblastoma development.Support or Funding InformationNICHD R00HD087430