Cell Stem Cell, published online 17 May 2012, doi:10.1016/j.stem.2012.03.001

Somatic cells can be reprogrammed into pluripotent cells called induced pluripotent stem cells by a combination of transcription factors, including Oct-4 and Sox2. Glucose conditions in culture also affect pluripotency, but a molecular explanation for this observation has been lacking. Now Jang et al. report that Oct-4 is modified with an O-linked N-acetylglucosamine (O-GlcNAc) in stem cells. Chemical or genetic inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc, reduced reprogramming efficiency as well as embryonic stem cell (ESC) maintenance in culture. The authors used succinylated wheat germ agglutinin and free GlcNAc to pull down and elute GlcNAc-modified proteins, which included Oct-4 and Sox2. Treatment with β-N-acetyl-hexosaminidase, an enzyme that removes O-linked glycans, confirmed the O linkage in Oct-4 and Sox2. Follow-on analyses revealed that Oct-4 was modified at Thr228 and that mutation of this site reduced reprogramming efficiency and ESC self-renewal. The authors also showed that chemical inhibition of OGA in differentiating ESCs, which lose O-GlcNAc on Oct-4, restored the Oct-4 modification and Oct-4–dependent reporter gene expression. Gene expression analysis and ChIP-seq, to reveal promoters occupied by and regulated by Oct-4, identified specific pluripotency genes whose expression was sensitive to the Oct-4 modification. Taken together, these data provide a direct molecular connection between O-GlcNAcylation and the activation of a pluripotency network.