84 / 2026-04-06 08:48:28

O-GlcNAcylation drives enhancer–promoter loop formation through phase separation and antagonizes loop extrusion

O-GlcNAcylation,loop extrusion,phase separation,enhancer–promoter loop

O-GlcNAcylation is a ubiquitous and dynamically reversible post-translational modification regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Previous studies have demonstrated that O-GlcNAcylation participates in diverse biological processes through the regulation of epigenetic modifications and gene expression. However, its contribution to chromatin remodeling and three-dimensional (3D) genome organization remains poorly defined. Here, we generated multi-omics datasets in C2C12 and C3H cells, including Hi-C, ChIA-PET, CUT&Tag, ATAC-seq, ACC-seq, and RNA-seq, to systematically investigate the regulatory role of O-GlcNAcylation in chromatin 3D interactions. We found that O-GlcNAcylation is significantly enriched in active chromatin regions, and its depletion leads to alterations in TADs, compartments, and loops, with the most pronounced changes observed in chromatin loops.

Mechanistically, O-GlcNAcylation exerts distinct regulatory effects on functional and structural loops. Its loss disrupts RNA Pol II-mediated loops while promoting the extension of CTCF-mediated loop extrusion, resulting in the formation of long range CTCF loops. These architectural changes are accompanied by widespread transcriptional dysregulation and drive cell fate toward apoptosis. Integration of proteomic data and motif enrichment analysis further revealed that O-GlcNAcylated proteins, including BRD4 and RFX1, are significantly enriched at differential Pol II loop anchors. Combined with CUT&Tag, ACC-seq, and functional assays, these findings suggest that O-GlcNAcylation enhances the chromatin binding capacity of key regulatory factors and promotes phase separation, thereby stabilizing chromatin loop structures. Collectively, our findings identify O-GlcNAcylation as a key regulator of 3D genome organization and provide mechanistic insight into how it modulates chromatin protein dynamics and phase separation to maintain Pol II loop stability, ultimately reshaping chromatin architecture and directing cell fate decisions.