Biomolecular condensates organize gene expression by concentrating proteins and nucleic acids into dynamic, membrane-less compartments through liquid-liquid phase separation (LLPS). Although post-translational modifications are increasingly recognized as important regulators of condensate behavior, whether intracellular O-GlcNAcylation follows a general principle in controlling phase separation has remained largely unknown.
In this study, the authors performed a proteome-wide analysis of the human O-GlcNAcome and discovered that O-GlcNAc sites are highly enriched within intrinsically disordered regions, where they frequently form densely clustered groups termed O-GlcNAc clusters. These clustered modifications are strongly associated with phase-separating proteins, particularly transcriptional regulators, suggesting a previously unrecognized molecular feature underlying transcriptional condensates.
Using BRD4 as a representative model, the authors identified twelve major O-GlcNAc sites within its C-terminal intrinsically disordered region and demonstrated that these sites act cooperatively to regulate condensate properties. Multi-omics analyses further revealed that O-GlcNAc-dependent remodeling of BRD4 condensates reshapes chromatin occupancy, transcriptional condensate assembly, and downstream gene expression programs controlling cell-cycle progression, DNA damage repair, and cell proliferation.
Key findings from the study include:

1. O-GlcNAc clusters are broadly associated with phase-separating proteins. O-GlcNAc sites frequently form clusters within intrinsically disordered regions, and proteins containing these clusters exhibit significantly higher phase-separation propensity. Similar regulatory effects were observed in multiple transcription-associated condensate proteins, supporting the general role of O-GlcNAc clusters in condensate regulation.
2. Clustered O-GlcNAcylation fine-tunes BRD4 phase separation. Multiple O-GlcNAc sites function cooperatively, rather than individually, to weaken BRD4 phase separation, resulting in smaller, more fluid condensates in vitro and in living cells.
3. O-GlcNAc-dependent regulation of BRD4 condensates controls transcription and cellular phenotypes. Reduced BRD4 O-GlcNAcylation enhances chromatin binding, particularly at active enhancers and super-enhancers, promotes co-condensation with transcriptional regulators, and activates gene programs involved in cell-cycle progression, DNA damage repair, and proliferation.

Together, these findings establish O-GlcNAc clusters as a new layer of the molecular grammar governing biomolecular condensates. The study provides a conceptual framework for understanding how multiple individually weak glycosylation events collectively regulate protein assembly, chromatin engagement, and gene expression, and suggests new opportunities for manipulating aberrant condensates in cancer and other diseases through precision O-GlcNAc editing.
DOI:10.1093/procel/pwag053