O-GlcNAc clusters in intrinsically disordered regions regulate transcriptional condensates and gene control

Post-translational modifications (PTMs) dynamically regulate the liquid-liquid phase separation (LLPS) that organizes biomolecular condensates, yet for the monosaccharide O-GlcNAc modification, the general rules and their links to protein function remain undefined. Here we identify O-GlcNAc as a widespread LLPS modulator by revealing clustered O-GlcNAc sites enriched in intrinsically disordered regions (IDRs) of transcription-related proteins, with BRD4 exhibiting this pattern most prominently. Twelve clustered O-GlcNAc sites are identified in BRD4 C-terminal IDR, where modifications decrease condensate size while increasing fluidity in vitro and in cells. Removing O-GlcNAc strengthened BRD4 binding at active enhancers and promoted LLPS-mediated recruitment of transcriptional cofactors, including YTHDC1, leading to elevated expression 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.