Molecular interactions underlying the phase separation of HP1α: Role of phosphorylation, ligand and nucleic acid binding

Abstract

Heterochromatin protein 1α (HP1α) is a crucial component for the proper maintenance of chromatin structure and function. It has been proposed that HP1α functions through liquid-liquid phase separation (LLPS), which allows it to sequester and compact chromatin into transcriptionally repressed heterochromatin regions. In vitro, HP1α can form phase separated liquid droplets upon phosphorylation of its N-terminus extension (NTE) and/or through interactions with DNA and chromatin. While it is known that LLPS requires homodimerization of HP1α and that it involves interactions between the positively charged hinge region of HP1α and the negatively charged phosphorylated NTE or nucleic acid, the precise molecular details of this process and its regulation are still unclear. Here, we combine computational modeling and experimental approaches to elucidate the phase separation properties of HP1α under phosphorylation-driven and DNA-driven LLPS conditions. We also tune these properties using peptides from four HP1α binding partners (Sgo1, CAF-1, LBR, and H3). In phosphorylation-driven LLPS, HP1α can exchange intradimer hinge-NTE interactions with interdimer contacts, which also leads to a structural change from a compacted to an extended HP1α dimer conformation. This process can be enhanced by the presence of positively charged peptide ligands such as Sgo1 and H3 and disrupted by the addition of negatively charged or neutral peptides such as LBR and CAF-1. In DNA-driven LLPS, both positively and negatively charged peptide ligands can perturb phase separation. Our findings demonstrate the importance of electrostatic interactions in the LLPS of HP1α where binding partners can modulate the overall charge of the droplets and screen or enhance hinge region interactions through specific and non-specific effects. Our study illuminates the complex molecular framework that can fine tune the properties of HP1α and that can contribute to heterochromatin regulation and function.

Phosphorylation regulates tau’s phase separation behavior and interactions with chromatin

“…For example, previous work has shown that negatively charged peptides can completely abolish LLPS of pHP1α and LLPS of HP1α and DNA. 47,79 While the effects of hyperphosphorylated tau in the nucleus are likely buffered by other components, our data indicate that the buildup of phosphorylation modifications on tau has the potential to disrupt the material properties of heterochromatin environments. Our observations are consistent with previous reports that pathological forms of tau lead to heterochromatin relaxation and loss of chromocenters.…”

“…79 Literature suggests that HP1α is phosphorylated by the CK2 kinase and that it is constitutively present in cells, pointing to its biological significance. 47,79,81 Suitably phosphorylated HP1α can also be produced recombinantly in E. coli using co-expression with the CK2. Upon combination of 50 μM tau and 50 μM pHP1α at low salt, the two instantly formed droplets, and 12mer arrays partitioned into these droplets when present ( Fig 6a ).…”

“…Unlike other electrostatically driven LLPS systems where high salt conditions abolish interactions, 4,47,79 the effect of salt on tau-12mer array samples is complex. Physiological concentrations of monovalent and divalent salts typically lead to a compact chromatin state, 61 which we also observe both in the presence of NaCl and MgCl2.…”

“…47,49,78 This LLPS process appears to be electrostatically driven and can be modulated by a number of HP1α binding partners. 47,49,51,53,[78][79][80] Tau's capacity to phase separate through an electrostatic mechanism and the observation that its depletion or pathology disrupts HP1α and heterochromatin clustering 20,31,33 prompts the question of a possible interplay between the phase separation of tau and HP1α. To explore this possibility, we reconstituted the heterochromatin environment in vitro by combining tau with phosphorylated HP1α (pHP1α, Fig.…”

“…We chose to work with HP1α phosphorylated on its N-terminal extension as this modification significantly reduces the saturation concentration required for LLPS in vitro in the absence of nucleic acids or chromatin from > 250 μM for unmodified HP1α to ~ 75 μM for pHP1α (concentrations compared at 75 mM KCl). 79 Literature suggests that HP1α is phosphorylated by the CK2 kinase and that it is constitutively present in cells, pointing to its biological significance. 47,79,81 Suitably phosphorylated HP1α can also be produced recombinantly in E. coli using co-expression with the CK2.…”