Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity, and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we use multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc-repressed genes. Finally, non-Hox Pc targets are more proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not induce frequent clustering of multiple distant Pc genes.
