The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs)^1,2,3,4. However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.

基因组在细胞核内折叠成三维结构的层次结构。在亚兆碱基尺度上，染色体形成拓扑关联域 (TAD) ^1,2,3,4。然而，TAD 在单细胞中如何折叠尚不清楚。在这里，我们揭示了使用超分辨率显微镜无法进行细胞群分析的 TAD 特征。TAD 结构和与其边界相关的物理隔离在各个细胞之间是可变的，但与大多数细胞中的相邻 TAD 相比，TAD 内的染色质混合更加丰富。TAD 的空间分离在细胞分化过程中进一步加剧。TAD 内有利的相互作用由粘连蛋白和 CTCF 通过不同的机制调节：粘连蛋白产生染色质接触和混合，而 CTCF 阻止 TAD 间接触。此外，TAD 被细分为离散的纳米结构域，这些纳米结构域持续存在于 CTCF 或粘连蛋白耗尽的细胞中，而核小体接触的破坏会改变其结构组织。总而言之，这些结果为纳米尺度上单个染色体的折叠提供了物理基础。