CAD-C reveals centromere pairing and near-perfect alignment of sister chromatids

Three-dimensional (3D) genome organization plays a central role in gene regulation, chromatin folding, and genome stability. Although chromosome-conformation capture (3C)-derived methods have revolutionized our understanding of genome architecture, most remain limited in resolution, in their capacity to detect multiway interactions and in their ability to distinguish sister chromatids. Here, we present CAD-C, a new chromatin-conformation capture strategy that uses Caspase-Activated DNase (CAD) to fragment chromatin. Fragmentation of chromatin to the nucleosome level by CAD digestion substantially enhances proximity ligation, enabling formation of multi-nucleosome ligation products. Nanopore sequencing of these long DNA molecules allows reconstruction of chromatin fiber connectivity and 3D contact maps with single nucleosome resolution. Importantly, CAD-C is able to identify sister-chromatid interactions at high resolution which reveals that centromeres are closely paired and that cohesin maintains sister chromatids in near perfect alignment where the same nucleosomes are associated across sisters. Such precise alignment has significant implications for chromosome structure and the mechanisms by which cohesion is established.