Revealing site-specific molecular features mediating contacts of heterochromatin protein 1 α (HP1α) and the interactions with chromatin and DNA in LLPS environments.

Serving as critical chromatin-binding effector proteins, the heterochromatin protein 1 (HP1) family possesses distinct biological functions, of which the mechanisms are unclear. Liquid-liquid phase separation (LLPS) of the HP1 family and their interaction with nucleosomes are crucial mechanisms governing chromatin activities. Here, we first investigate phosphorylated HP1α (pHP1α) LLPS by NMR spectroscopy, confocal microscopy, and molecular dynamics simulations. This reveals that the phosphorylated N-terminal extension (NTE) and hinge region are key determinants of pHP1α LLPS. We next explore the interactions between pHP1α and telomeric nucleosome arrays and a relatively more open Widom 601 nucleosome array under LLPS conditions. The results suggest that the interaction of pHP1α and nucleosomes is driven by the self-association of pHP1α mediated by intermolecular interaction between the NTE and hinge regions and the specific binding of the pHP1α chromo domain (CD) to the N-terminal histone tail of H3K9me3 in the nucleosomes. In addition, experiments elucidate that DNA can mediate the pHP1α LLPS by competing with the NTE to interact with hinge regions. Together, our findings provide atomic-level insights into the determinants of the molecular features driving the self-association of the pHP1α and the interactions with DNA and nucleosomes.