OTX2's Dual Mode of Degradation is Regulated by O‐GlcNAcylation
Authors: Wulff Fuentes Eugenia A. , Boakye Jeffrey , Pereckas Michael , Berendt Rex , Hanover John A. , Olivier‐Van Stichelen Stephanie 
Published: May 2022

The O‐GlcNAc post‐translational modification is reversibly added onto intracellular proteins. It is a unique glucose rheostat for cell signaling relying on the availability of UDP‐GlcNAc, itself reflecting extracellular glucose. With more than 7000 human targets identified to date, O‐GlcNAcylation regulates numerous physiological processes such as cell cycle, transcriptional/translational regulation, protein localization or degradation, and development. Therefore, O‐GlcNAcylation is a molecular bridge between dietary glucose level and proper signaling regulation.Using cellular and mouse models, our lab has previously delved into the consequences of hyper‐O‐GlcNAcylation in the brain. Among phenotypes of obesity and growth defects, the pituitary gland of these mice was generally delayed in development. A critical aspect of pituitary’s ontogeny is the transient expression of the homeobox protein OTX2, an O‐GlcNAcylated protein. However, the function of O‐GlcNAcylation in regulating OTX2 has not been investigated. Interestingly, in hyper‐O‐GlcNAcylated mouse embryonic stem cells, this transcription factor’s expression increased, suggesting that O‐GlcNAc plays a role in OTX2 stability/degradation. Thus, we hypothesized that OTX2 is degraded by the proteasome and O‐GlcNAc cycling regulates its timely degradation.Using a combination of proteasome inhibition (MG‐132 mediated) and O‐GlcNAc increase (Thiamet‐G, TG), we demonstrated that endogenous OTX2 was indeed degraded by the proteasome. Interestingly, increased O‐GlcNAc levels caused further stabilization of OTX2, additional to the one achieved with proteasome inhibition alone. This suggested that OTX2 utilizes another degradation pathway, possibly autophagy. We showed that the macroautophagy inhibitor Chloroquine (CQ) prevented OTX2 degradation, and the addition of TG did not further stabilize the protein. This confirmed that OTX2 has two degradation modes, and that O‐GlcNAcylation is involved in the autophagy‐mediated degradation of this protein. However, we believe that autophagic degradation of OTX2 only occurs when this homeobox protein is abnormally overexpressed, such as in Medulloblastoma. Similar crosstalk between proteasome and autophagy has been demonstrated by others for developmental transcription factors like SOX2 and OCT4, following proteasome inhibition. We performed mass spectrometry site‐mapping of OTX2, and identified three O‐GlcNAc sites in the central domain of OTX2, likely involved in its autophagy‐mediated degradation.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 foresee that O‐GlcNAcylated OTX2 may play a major role in Medulloblastoma pathogenesis.