Loss of SUMOylation drives aberrant PRC1 clustering and 3D genome rewiring independent of H3K27me3.
Polycomb repressive complex 1 (PRC1) forms nuclear condensates that organize target chromatin domains. SUMOylation modulates PRC1 clustering, but its impact on condensate properties and 3D genome architecture remains unclear. Here, we show that depletion of small ubiquitin-like modifier (SUMO) in Drosophila wing imaginal discs transforms PRC1 condensates into large structures with reduced molecular dynamics. Biophysical modeling suggests that the changes in PRC1 self-interactions are responsible for the formation of large PRC1 condensates when SUMO is depleted. Interestingly, this biophysical reorganization occurs without global loss of the H3K27me3 mark. Instead, Hi-C reveals widespread rewiring of topologically associating domain (TAD) interactions. PRC1-bound TADs lose specific long-range contacts with each other while gaining ectopic interactions with active chromatin. These topological shifts correlate with gene misregulation independently of changes in Polycomb histone modifications. Our results establish SUMOylation as a critical regulator of PRC1 condensates, demonstrating that post-translational control of biomolecular condensation modulates 3D genome architecture and transcriptional output through mechanisms separable from histone mark deposition.