DNA supercoiling is a biophysical feature of the double helix with a pivotal role in biological processes. However, understanding of DNA supercoiling in the chromatin remains limited. Here, we developed azide-trimethylpsoralen sequencing (ATMP-seq), a DNA supercoiling assay offering quantitative accuracy while minimizing genomic bias and background noise. Using ATMP-seq, we directly visualized transcription-dependent negative and positive twin-supercoiled domains around genes and mapped kilobase-resolution DNA supercoiling throughout the human genome. Remarkably, we discovered megabase-scale supercoiling domains (SDs) across all chromosomes that are modulated mainly by topoisomerases I and IIβ. Transcription activities, but not the consequent supercoiling accumulation in the local region, contribute to SD formation, indicating the long-range propagation of transcription-generated supercoiling. Genome-wide SDs colocalize with A/B compartments in both human and Drosophila cells but are distinct from topologically associating domains (TADs), with negative supercoiling accumulation at TAD boundaries. Furthermore, genome-wide DNA supercoiling varies between cell states and types and regulates human gene expression, underscoring the importance of supercoiling dynamics in chromatin regulation and function.

DNA 超螺旋是双螺旋的生物物理特征，在生物过程中发挥着关键作用。然而，对染色质 DNA 超螺旋的了解仍然有限。在此，我们开发了叠氮化物三甲基补骨脂素测序 (ATMP-seq)，这是一种 DNA 超螺旋检测方法，可提供定量准确性，同时最大限度地减少基因组偏差和背景噪音。使用 ATMP-seq，我们直接可视化基因周围转录依赖性负双超螺旋结构域和正双超螺旋结构域，并绘制了整个人类基因组中千碱基分辨率的 DNA 超螺旋图。值得注意的是，我们在所有染色体上发现了主要由拓扑异构酶 I 和 IIβ 调节的兆碱基级超螺旋结构域 (SD)。转录活动，而不是随后在局部区域的超螺旋积累，有助于SD的形成，表明转录产生的超螺旋的远距离传播。全基因组范围的 SD 与人类和果蝇细胞中的 A/B 区室共定位，但与拓扑关联域 (TAD) 不同，在 TAD 边界处具有负超螺旋积累。此外，全基因组 DNA 超螺旋因细胞状态和类型而异，并调节人类基因表达，强调了超螺旋动力学在染色质调节和功能中的重要性。