BRD4 O-GlcNAcylation attenuates phase separation to regulate transcription

BRD4 undergoes liquid-liquid phase separation (LLPS) at super-enhancers (SEs) to facilitate gene control. However, the regulatory mechanisms and transcriptional consequences of BRD4 condensation remain elusive. Here, we show that the BRD4 C-terminal intrinsically disordered region (IDR) is a primary interacting region for O-GlcNAc transferase (OGT), enabling O-GlcNAc modifications at 12 potential sites. O-GlcNAcylation attenuates BRD4 LLPS, leading to small puncta and increased fluidity, which significantly declines occupancy and transcription at SEs. We further demonstrate that loss of O-GlcNAcylation on BRD4 led to elevated gene expression relating to cell cycle and DNA damage, allowing cells less sensitive to DNA damage and promoting faster growth. Therefore, we identify an O-GlcNAc–mediated regulatory mechanism for BRD4 LLPS that governs gene control at SEs, showcasing how O-GlcNAc modifications can influence transcriptional activities at SEs through a phase separation model.