Three-dimensional (3D) genome folding has a fundamental role in 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 CRE 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 (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and 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.

三维 （3D） 基因组折叠通过促进或限制顺式调节元件 （CRE） 之间的染色质相互作用，在发育基因的调控中起着重要作用。多梳反应元件 （PREs） 是一种特定类型的 CRE，参与黑腹果蝇转录状态的记忆。PRE 充当多梳组 （PcG） 蛋白的成核位点，该蛋白沉积抑制组蛋白标记 H3K27me3，导致形成一类称为多梳结构域的拓扑关联结构域 （TAD）。PREs 可以建立循环接触，在发育过程中稳定关键发育基因的基因抑制。然而，PRE 环微调基因表达的机制尚不清楚。使用成簇的规则间隔短回文重复序列和 Cas9 基因组工程，我们专门扰乱了 PRE 接触或增强子功能，并使用包括 4C-seq、Hi-C 和 Hi-M 在内的互补方法来分析染色质结构扰动如何影响基因表达。我们的结果表明，dac 基因位点的 PRE 环在果蝇发育过程中充当组成型 3D 染色质支架，其形成独立于基因表达状态并具有多种功能;它限制了增强子-启动子通讯，并有助于增强子特异性。