In vitro reconstitution of chromatin domains [ChIP-Seq]

A key step towards defining the structure-function relationship of the genome is to identify the molecular mechanisms that drive higher-order genome folding. To this end, we reconstituted five S. cerevisiae chromosomes in vitro and developed a high-resolution MNase-based chromosome conformation capture assay to measure their 3D organization. We show that the formation of regularly spaced and phased nucleosome arrays is sufficient to drive higher-order genome folding into domains that resemble in vivo genome organization and thereby demonstrate that neither loop extrusion nor transcription are required for domain formation. The domain boundaries correspond to nucleosome-free regions and insulation strength scales with their width. Integrated molecular dynamics simulations show that domain compaction is dependent on nucleosome linker length, with longer linkers forming more compact structures. Together, our work demonstrates that fundamental properties of chromatin fibers are important determinants of higher-order genome folding and provides a proof-of-principle for bottom-up 3D genome studies. We used a genome-wide in vitro reconstitution system (Oberbeckmann et al. 2021) to study how nucleosome positioning affects higher-order genome folding. To this end, we established a method to map 3D nucleosome contacts in vitro and named it in vitro Micro-C. DNA sequence of 5 chromosomes (V-IX) from S. cerevisiae was reconstituted into chromatin by salt gradient dialysis and then incubated with transcription factors only or additionally with various remodeler. Nucleosome positioning was confirmed with MNase-seq, while higher-order nucleoosme contacts were mapped with in vitro Micro-C.