Elevated O-GlcNAc Levels Activate Epigenetically Repressed Genes and Delay Mouse ESC Differentiation Without Affecting Naïve to Primed Cell Transition

Abstract

The differentiation of mouse embryonic stem cells (ESCs) is controlled by the interaction of multiple signaling pathways, typically mediated by post‐translational protein modifications. The addition of O‐linked N‐acetylglucosamine (O‐GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins is one such modification (O‐GlcNAcylation), whose function in ESCs is only now beginning to be elucidated. Here, we demonstrate that the specific inhibition of O‐GlcNAc hydrolase (Oga) causes increased levels of protein O‐GlcNAcylation and impairs differentiation of mouse ESCs both in serum‐free monolayer and in embryoid bodies (EBs). Use of reporter cell lines demonstrates that Oga inhibition leads to a reduction in the number of Sox1‐expressing neural progenitors generated following induction of neural differentiation as well as maintained expression of the ESC marker Oct4 (Pou5f1). In EBs, expression of mesodermal and endodermal markers is also delayed. However, the transition of naïve cells to primed pluripotency indicated by Rex1 (Zfp42), Nanog, Esrrb, and Dppa3 downregulation and Fgf5 upregulation remains unchanged. Finally, we demonstrate that increased O‐GlcNAcylation results in upregulation of genes normally epigenetically silenced in ESCs, supporting the emerging role for this protein modification in the regulation of histone modifications and DNA methylation. Stem Cells 2014;32:2605–2615

Embryonic Stem Cell Proliferation Stimulated By Altered Anabolic Metabolism From Glucose Transporter 2-Transported Glucosamine

“…Our findings differ from those of some groups that reported that O -GlcNAcylation of pluripotency or cell cycle regulatory factors stimulates proliferation or pluripotency and inhibits differentiation of ESC12131436. We found that exogenous GlcN only stimulated proliferation of LG-ESC and did not stimulate pluripotency nor inhibit differentiation, and, while GlcN did increase total protein O -GlcNAcylation, stimulation of proliferation was not due to increased O -GlcNAcylation.…”

Metabolism in pluripotency: Both driver and passenger?

“…Additionally, as previously mentioned, HBP couples macromolecular metabolism to O-GlcNAc modifications in proteins, including epigenetic modifiers (Figure 3) (94). Cycling of these posttranslational modifications by OGT and OGA are required for naïve and primed state induction, maintenance, and differentiation (95,96). OGT interacts with TET1 enzymes preferentially at transcription start sites, thereby influencing gene expression (58).…”

Chemical Modulation of Protein O-GlcNAcylation via OGT Inhibition Promotes Human Neural Cell Differentiation

“…9,20–22 Liu et al reported higher levels of O-GlcNAc, OGT, and OGA in neurons compared to non-neuronal cells in the rat brain. 23 Maintaining high levels of O-GlcNAcylation prevents ectodermal differentiation of mouse ESCs, 13 impairs axonal branching, 24 and inhibits proteasome function. 25 A recent study using human embryonic stem cells (hESCs) found that excess O-GlcNAc decreased the expression of neural markers PAX6 and SOX1.…”