Multiplexed chromatin imaging reveals predominantly pairwise long-range coordination between Drosophila Polycomb genes

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. [Display omitted] •3D proximity between Polycomb targets is rare in Drosophila embryos•Clustering of Pc genes involves in most cases only two targets•3D proximity of Pc targets depends on segment-specific expression Gurgo et al. explore how Polycomb proteins, crucial for cell identity and development, interact in 3D space. The findings reveal that these interactions are rare, specific, and regulated during development. This enhances our understanding of gene regulation, potentially impacting fields like developmental biology and disease research.