O-GlcNAc clusters attenuate BRD4 phase separation for transcription regulation

Abstract

Post-translational modifications (PTMs) regulate the liquid-liquid phase separation (LLPS) that organizes biomolecular condensates essential for transcription, yet for the monosaccharide O-GlcNAc, the general rules and their links to protein function remain undefined, with evidence largely limited to isolated cases rather than systematic analyses. Here we combine proteome-scale analyses with experiments to show that O-GlcNAc is a widespread LLPS modulator: glycosites are enriched in intrinsically disordered regions and form dense O-GlcNAc clusters. Using BRD4 as a model, we identified twelve clustered sites in its C-terminal IDR and showed that O-GlcNAc decreases condensate size while increasing fluidity in vitro and in cells. Removing O-GlcNAc strengthened BRD4 binding at active enhancers and promoted LLPS-mediated co-recruitment of transcriptional factors, including YTHDC1, leading to altered expression programs linked to the cell cycle and DNA repair. Our findings define O-GlcNAc clusters as regulators of condensate material properties and transcriptional outcomes, supporting a general paradigm in which PTMs fine-tune the molecular grammar of biomolecular condensates.