Isolating the Effect of Nucleosome Modification Driven Phase Separation on Chromatin Organization.

Several mechanisms regulate the three-dimensional organization of the genome. Though motor protein-driven loop extrusion has been studied extensively, the role of phase separation in establishing chromatin structure remains unclear. In this study, we aim to determine how histone modifications and variants affect nucleosome phase separation and drive chromatin organization in the absence of loop extrusion and other such mechanisms. Using Flory-Huggins theory and the mixing rule, we calculate the contact energy between nucleosome types from condensability scores obtained from in vitro condense-seq experiments. We run coarse-grained polymer simulations of chromatin with these contact energies and compare the resulting contact maps with experimental Hi-C maps of the same region. At the 10-20 Mb scale, simulations with contact energies inferred from polycation condense-seq recreate experimentally observed A/B compartment profiles. This suggests that heterochromatin formation at the compartment scale is regulated primarily by nucleosomal phase separation in the absence of other proteins. Future condense-seq experiments can be performed with various condensing agents besides polycations, including proteins such as HP1α. Our approach will provide insight into how these factors regulate chromatin architecture via phase separation. Disentangling the various mechanisms contributing to genome organization will enable us to more reliably predict and control gene expression, with applications in medicine and synthetic biology.