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，在发育基因的调节中发挥重要作用。多梳反应元件 (PRE) 是一种特殊的 CRE，参与果蝇转录状态的记忆。 PRE 作为 Polycomb 族 (PcG) 蛋白的成核位点，沉积抑制性组蛋白标记 H3K27me3，从而形成一类称为 Polycomb 结构域的拓扑关联结构域 (TAD)。 PRE 可以建立循环接触，稳定发育过程中关键发育基因的基因抑制。然而，PRE 循环微调基因表达的机制尚不清楚。使用成簇规则间隔的短回文重复序列和 Cas9 基因组工程，我们专门扰乱 PRE 接触或增强子功能，并使用包括 4C-seq、Hi-C 和 Hi-M 在内的补充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基因座上的 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，并且具有多种功能。它限制增强子与启动子之间的交流并有助于增强子的特异性。