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 the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function: it restricts enhancer promoter communication and contributes to enhancer specificity.
Overall design
Hi-C experiments of wilt type imaginal leg discs and pupae or imaginal leg discs carrying a gypsy insertion and/or enhancer deletion 4C-seq experiments of wilt type embryos, imaginal leg discs or pupae or embryos, imaginal leg discs or pupae carrying a gypsy insertion and/or enhancer deletion Cut&Run experiments of wilt type imaginal leg discs or imaginal leg discs carrying a gypsy insertion and/or enhancer deletion
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