Credit: Vicky Sumersby/NPG

O-linked β-d-N-acetylglucosamine (O-GlcNAc) modification has recently emerged as a key regulator of cell physiology, controlling aspects such as nutrient sensing and cell cycle progression. Youn and colleagues reveal that this is not restricted to differentiated cells; O-GlcNAcylation also regulates embryonic stem (ES) cell pluripotency.

Previous studies suggested that ES cell maintenance may be regulated by O-GlcNAcylation, in particular by reporting that the levels of glucose in medium affect whether ES cells differentiate or self-renew. Consistent with these observations, the authors found that loss of O-GlcNAc transferase (OGT; which mediates O-GlcNAcylation) led to reduced proliferation and self-renewal of ES cells as well as decreased reprogramming efficiency of induced pluripotent stem (iPS) cells. By contrast, overexpression of OGT or depletion of O-GlcNAcase (OGA; which removes O-GlcNAc) increased reprogramming efficiency and decreased differentiation, respectively. These findings indicate that O-GlcNAcylation promotes ES cell self-renewal and inhibits differentiation.

ES cell self-renewal is regulated by a pluripotency network comprising OCT4 (also known as POU5F1 and OCT3), SOX2, KLF4 (Krüppel-like factor 4) and NANOG, so the authors hypothesized that O-GlcNAc may be modifying some of these factors. Indeed, OCT4 and SOX2 were O-GlcNAcylated, and this modification decreased following ES cell differentiation. Moreover, OCT4 O-GlcNAcylation depended on the OCT4 residue Thr228, as mutation of this residue decreased O-GlcNAc levels in ES cells. Importantly, mutation of OCT4 at Thr228 resulted in reduced reprogramming efficiency of mouse embryonic fibroblasts and defective self-renewal of ES cells, which suggests that O-GlcNAcylation of OCT4 is required to maintain pluripotency.

But how is this achieved? Because OCT4 O-GlcNAcylation did not affect protein stability or cell localization, the authors postulated that it may regulate OCT4 activity as a transcription factor. Using a luciferase reporter assay, the authors confirmed that O-GlcNAcylation increases the transcriptional activity of OCT4. Further analysis indicated that O-GlcNAcylated OCT4 directly regulates the transcription of 29 genes, including pluripotency-related genes such as Nanog and Klf4.

Together, these findings reveal that OCT4 O-GlcNAcylation acts as a key regulator of pluripotency, adjusting the transcription of pluripotency-related genes according to external signals.