O-GlcNAcylation of Sox2 at threonine 258 regulates the self-renewal and early cell fate of embryonic stem cells

Dong Keon Kim,Hye Jin You,In-Young Hwang,Hyun Mu Shin,Hansol Jang,Jang-Seok Lee,Ji-Woong Choi,Hyonchol Jang,Hee Yeon Kim,Suji Han,Eun Young Lee,Hong-Duk Youn

doi:10.1038/s12276-021-00707-7

Dong Keon Kim, Hye Jin You + Show 10 more

Open Access

https://doi.org/10.1038/s12276-021-00707-7

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Abstract

Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.

Highlights

O-GlcNAcylation, which entails attachment of a single monosaccharide, N-acetyl-D-glucosamine (GlcNAc), to a serine or threonine residue of a nucleocytoplasmic protein via an Oβ-glycosidic linkage, is associated with the sensing of nutrients[1,2] and affects embryonic stem cell (ESC) pluripotency[3,4,5]
The cells infected with Sox[2] T258A produced significantly fewer APpositive colonies than those infected with WT Sox[2], whereas the cells infected with Sox[2] S248A and S248D did not (Fig. 1b)
Because retroviral promoters are commonly silenced during long-term ESC culture[38], gene expression using retroviruses is inadequate for observing mid- to long-term effects of Sox[2] mutation

Summary

Introduction

O-GlcNAcylation, which entails attachment of a single monosaccharide, N-acetyl-D-glucosamine (GlcNAc), to a serine or threonine residue of a nucleocytoplasmic protein via an Oβ-glycosidic linkage, is associated with the sensing of nutrients[1,2] and affects embryonic stem cell (ESC) pluripotency[3,4,5]. Decreased expression of Ogt leads to downregulation of the global O-GlcNAc level, which in turn was shown to reduce mouse ESC self-renewal and the efficiency of reprogramming mouse embryonic fibroblasts (MEFs) to induce pluripotent stem cells (iPSCs)[3]. Ogt decreased global O-GlcNAc levels and accelerated the differentiation of human ESCs6. Upregulation of global O-GlcNAc levels, either via enhanced Ogt expression or glucose levels in the culture medium, increased the efficiency of reprogramming MEFs into iPSCs3. Upregulation of global O-GlcNAc levels via chemical inhibition of Oga suppressed mouse ESC differentiation[7]

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