Project Summary The formation and dissolution of heterochromatin allows for different genes to be silenced or upregulated, and can result in different cell types forming from the same genetic material. Improper function of heterochromatin has been linked to the progression of many forms of cancer, and recent work has also uncovered links to Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). Heterochromatin function emerges in part from the ability of the heterochromatic proteins (HP1) to recruits ligands and spread across the genome. In humans, three members of the HP1 family have been identified, which can phase separate into liquid-like proteinaceous droplets in vitro. The three proteins, while similar, display different responses to phosphorylation, by either being more prone to phase separate or less. These HP1 proteins also interact preferentially with methyl-binding proteins (MBDs), which are known to associate with particular regions of the genome. The molecular details of heterochromatin-related proteins have yet to be characterized due to the number of components, the disordered nature of many of the components, and the wide range of relevant length scales over which the processes of seeding and dissolution occur. This project will apply a combination of computational and theoretical techniques, backed by experimental collaborations to provide a mechanistic understanding of the driving forces underlying heterochromatin function and dysfunction by 1) identifying the interaction modes relevant to HP1 and MBD phase separation at the atomic and molecular level 2) quantifying the degree to which different proteins and ligands incorporate into multicomponent assemblies/condensates 3) determining the extent to which post-translational modifications perturb phase separation, and 4) characterize the incorporation of nucleic acids into protein-rich condensates, determining the effects of DNA hybridization and RNA on incorporation and dynamics within condensates. These approaches will generate detailed molecular-level information on the assembly of many components into heterochromatin assemblies, and provide the background knowledge needed for better understanding its regulation, and disease relevance.