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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. [ABSTRACT FROM AUTHOR]

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG. Per-O-acetylated unnatural monosaccharides are popular tools for glycan labeling in live cells but can undergo unwanted side reactions with cysteines. Here, the authors develop unnatural sugars in a partially esterified form that are inert towards cysteines, and use them to probe O-GlcNAcylation in mESCs. [ABSTRACT FROM AUTHOR]