Project Summary / Abstract Appropriate gene expression underlies every aspect of cellular biology from viral replication to multicellular organismal development to cancer. A key aspect of appropriate gene expression is the division of the genome into heterochromatin, a more tightly compacted and transcriptionally silent portion of the genome, and euchromatin, a more open, transcriptionally active portion. Euchromatic regions are thought to be defined by the constant activity of sequence-specific transcription, but the interactions that define and maintain heterochromatin are still an outstanding question in the field. Heterochromatin Protein 1α (HP1α) is a major structural component of constitutive heterochromatin and is thought to mediate chromatin compaction. Recent studies show that recombinantly purified HP1α undergoes liquid-liquid demixing on the addition of DNA to form distinct protein-rich and protein-poor phases. Similarly, N-terminally phosphorylated HP1α (nPhos HP1α) spontaneously demixes even in the absence of DNA. This phase separating ability has suggested new potential mechanisms for heterochromatin function. In particular, the differential chromatin affinities and phase separating abilities of nPhos and unmodified HP1α suggest that N-terminal phosphorylation may allow the cell to tune its heterochromatin compartment. However, more work on nPhos HP1α biochemistry is required prior to make targeted models of in vivo function. The goal of this proposal is to determine the properties of nPhos HP1α available to the cell for the protection and sequestration of heterochromatin. I will measure the strength and kinds of interaction between nPhos and unmodified HP1α, and whether the two species form miscible phases in vitro using light and fluorescence microscopy. I will also measure the viscoelasticity of and the diffusion of molecules within these phases through correlated fluorescence fluctuations and micro-rheology to determine whether solvated molecules can freely exchange between nPhos / HP1α phases. I will then correlate the physical material properties of pure and mixed HP1α phases with measurements of chemical environment and heterochromatin-associated enzymatic activity within HP1α droplets using established enzymatic assays as well as fluorescent pH, salt, redox, and hydrophobicity probes. Lastly, I will determine the mesoscale structure of nPhos and HP1α droplets containing heterochromatin-associated ligands using Soft X-ray Tomography (SXT). By generating HP1α droplets in various heterochromatin contexts (chromatin, RNAi, known protein binding partners) I will be able to determine how HP1α droplet structure is regulated, and how it relates to the corresponding material and chemical properties. These studies will provide a comprehensive characterization of nPhos HP1α behavior, and permit generation of targeted models for nPhos HP1α function for in vivo testing.