A nanomolar-affinity antibody enabling sensitive labeling and high-resolution genomic mapping of O-GlcNAc

High-affinity O-linked β-N-acetylglucosamine (O-GlcNAc)-specific antibodies are crucial for the sensitive detection and functional study of O-GlcNAcylation. Here, we report the development of a nanomolar-affinity O-GlcNAc antibody named EPR19847 by using a degenerate O-GlcNAcylated peptide as the immunogen. The EPR19847 antibody can be used for O-GlcNAc detection, imaging, isolation, and chromatin immunoprecipitation. Using this antibody, O-GlcNAcylated proteins are enriched and identified in mouse embryonic stem cells (mESCs). Chromatin immunoprecipitation followed by next-generation sequencing demonstrates that O-GlcNAc is enriched around the promoters of highly expressed genes, and that its genomic distribution is positively correlated with transcription-activating marks including H3K4me3, H3K9ac, and RNA polymerase II in mESCs. Further analysis suggests a high occupancy of O-GlcNAcylation within super-enhancers. We anticipate that EPR19847 will serve as a powerful tool for the highly sensitive detection and systematic functional dissection of O-GlcNAc. O‑GlcNAcylation is a widespread protein modification, but sensitive tools for its detection have been limited. Here, the authors develop a high‑affinity O‑GlcNAc antibody that enables detection, enrichment and genomic mapping of O‑GlcNAcylated proteins, revealing associations with active promoters and super‑enhancers.