In vitro reconstitution of chromatin domains

The spatial organization of the genome modulates nuclear processes, including transcription, replication, and DNA repair1,2. Eukaryotic genomes are organized into distinct 3D chromatin domains3. However, the molecular mechanisms that drive the formation of these domains are difficult to dissect in vivo and remain poorly understood. Here, we reconstitute S. cerevisiae chromatin in vitro and determine its 3D organization at sub-nucleosome resolution by MNase-based chromosome conformation capture and molecular dynamics simulations. We show that regularly spaced and phased nucleosome arrays form chromatin domains in vitro that resemble domains in vivo. This demonstrates that neither loop extrusion nor transcription are required for domain formation. In addition, we find that the boundaries of reconstituted domains correspond to nucleosome-free regions and that insulation strength scales with their width. Finally, we show that domain compaction depends on nucleosome linker length, with longer linkers forming more compact structures. Together, our results demonstrate that nucleosome positioning is sufficient to reconstitute chromatin domains and provide a proof-of-principle for bottom-up 3D genome studies.