PRE loops constitute a topological chromatin structure that restricts and specifies enhancer promoter communication

3D genome folding plays a fundamental role for the regulation of developmental genes by facilitating or constraining chromatin interactions between cis regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CREs involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TADs) called Polycomb domains. PREs can establish looping contacts that stabilize gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine tune gene expression is unknown. Using CRISPR/Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C, and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that PRE loops constitute a landmark topological structure during Drosophila development. These loops form independently of gene expression states and have a versatile function: they restrict enhancer promoter communication and they contribute to enhancer specificity.