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
Highlights
Over the last decade there have been major advances in our understanding of the mechanisms controlling embryonic stem cell (ESC) behavior in response to the changing extracellular environment [1]
We sought to determine whether the O-GlcNAc levels in ESCs can be modulated by inhibiting the O-GlcNAc hydrolase (Oga) enzyme activity that removes O-GlcNAc using GlcNAcstatin C (GNS), a highly potent and specific small molecule Oga inhibitor that exhibits 164fold selectivity for Oga when tested against the closely related lysosomal hexosaminidases HexA/B [28] and a competitive inhibitor of the O-GlcNAc transferase Ogt (4Ac5SGlcNAc) [29]
Treatment of ESCs with GNS resulted in a dose-dependent increase in cellular O-GlcNAc levels (Fig. 1C) including O-GlcNAcylation of the transcription factor Sox2 on S248 (Fig. 1D), with no discernable effects on cell viability or proliferation even after prolonged treatment (Fig. 1E, 1F)
Summary
Over the last decade there have been major advances in our understanding of the mechanisms controlling embryonic stem cell (ESC) behavior in response to the changing extracellular environment [1]. These mechanisms largely involve engagement of signal transduction relays that operate by post-translational modifications of proteins. Signaling mediated by Erk1/2 target phosphorylation has recently been implicated in regulating the transition between these two states and initiation of differentiation [2]. Reversible protein modification by addition of O-linked Nacetylglucosamine to serine or threonine residues (O-GlcNAcylation) was first described 30 years ago [3] and occurs with similar time scales, dynamics, and stoichiometry as protein phosphorylation, with which it sometimes competes. Its addition and removal are catalyzed by one transferase (Ogt) and one hydrolase (Oga; known as Mgea5), respectively
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