Although enhancers are central regulators of mammalian gene expression, the mechanisms underlying enhancer–promoter (E-P) interactions remain unclear. Chromosome conformation capture (3C) methods effectively capture large-scale three-dimensional (3D) genome structure but struggle to achieve the depth necessary to resolve fine-scale E-P interactions. Here, we develop Region Capture Micro-C (RCMC) by combining micrococcal nuclease (MNase)-based 3C with a tiling region-capture approach and generate the deepest 3D genome maps reported with only modest sequencing. By applying RCMC in mouse embryonic stem cells and reaching the genome-wide equivalent of ~317 billion unique contacts, RCMC reveals previously unresolvable patterns of highly nested and focal 3D interactions, which we term microcompartments. Microcompartments frequently connect enhancers and promoters, and although loss of loop extrusion and inhibition of transcription disrupts some microcompartments, most are largely unaffected. We therefore propose that many E-P interactions form through a compartmentalization mechanism, which may partially explain why acute cohesin depletion only modestly affects global gene expression.

尽管增强子是哺乳动物基因表达的核心调节因子，但增强子-启动子（EP）相互作用的机制仍不清楚。染色体构象捕获 (3C) 方法可以有效捕获大规模三维 (3D) 基因组结构，但难以达到解析精细 EP 相互作用所需的深度。在这里，我们通过将基于微球菌核酸酶 (MNase) 的 3C 与平铺区域捕获方法相结合来开发区域捕获 Micro-C (RCMC)，并仅通过适度测序即可生成报告的最深的 3D 基因组图谱。通过在小鼠胚胎干细胞中应用 RCMC，并达到约 3170 亿个独特接触的全基因组等效值，RCMC 揭示了以前无法解析的高度嵌套和焦点 3D 相互作用的模式，我们将其称为微区室。微区室经常连接增强子和启动子，尽管环挤出的丧失和转录的抑制破坏了一些微区室，但大多数微区室基本上不受影响。因此，我们提出许多 EP 相互作用是通过区室化机制形成的，这可能部分解释了为什么急性粘连蛋白耗竭仅适度影响全局基因表达。