Chemoproteomic and Transcriptomic Analysis Reveals that O‐GlcNAc Regulates Mouse Embryonic Stem Cell Fate through the Pluripotency Network

Many pluripotency transcription factors (PTFs) were found to be O-GlcNAcylated in mouse embryonic stem cells (mESCs) and the O-GlcNAcylation stoichiometry at various modification sites suppressed during differentiation. Mechanistically, O-GlcNAc acts at the PTF network level and inhibition of O-GlcNAcylation enhances the expression of neuronal lineage-specific genes, promoting mESC-derived neuronal differentiation.

Abstract

Self-renewal and differentiation of embryonic stem cells (ESCs) are influenced by protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification, but the underlying mechanism remains incompletely understood. Herein, we report the identification of 979 O-GlcNAcylated proteins and 1340 modification sites in mouse ESCs (mESCs) by using a chemoproteomics method. In addition to OCT4 and SOX2, the third core pluripotency transcription factor (PTF) NANOG was found to be modified and functionally regulated by O-GlcNAc. Upon differentiation along the neuronal lineage, the O-GlcNAc stoichiometry at 123 sites of 83 proteins—several of which were PTFs—was found to decline. Transcriptomic profiling reveals 2456 differentially expressed genes responsive to OGT inhibition during differentiation, of which 901 are target genes of core PTFs. By acting on the core PTF network, suppression of O-GlcNAcylation upregulates neuron-related genes, thus contributing to mESC fate determination.

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