Heterochromatin Protein HP1α Gelation Dynamics Revealed by Solid‐State NMR Spectroscopy

Abstract

Heterochromatin protein 1α (HP1α) undergoes liquid–liquid phase separation (LLPS) and forms liquid droplets and gels in vitro, properties that also appear to be central to its biological function in heterochromatin compaction and regulation. Here we use solid‐state NMR spectroscopy to track the conformational dynamics of phosphorylated HP1α during its transformation from the liquid to the gel state. Using experiments designed to probe distinct dynamic modes, we identify regions with varying mobilities within HP1α molecules and show that specific serine residues uniquely contribute to gel formation. The addition of chromatin disturbs the gelation process while preserving the conformational dynamics within individual bulk HP1α molecules. Our study provides a glimpse into the dynamic architecture of dense HP1α phases and showcases the potential of solid‐state NMR to detect an elusive biophysical regime of phase separating biomolecules.

On the relations of phase separation and Hi-C maps to epigenetics

“…On the face of it, the three phase-separated HP1α subcompartments appear to correspond with the three kinetic fractions observed in FRAP studies. However, a notable difference is that mammalian HP1α liquid droplets form in vitro independently of chromatin [25,27] while the 'slow' fraction is dependent upon the in vivo interaction of HP1 with its ligand, H3K9me3 [55,57]. Moreover, the CasDrop system has shown that mammalian HP1α is unlikely to form liquid droplets in vivo [69].…”

“…They represent a potential link between compartmentalization and epigenetics that will be explored in this paper. We now turn to these hallmarks with a focus on mammalian HP1 proteins because recent in vitro work has indicated that they form liquid-liquid condensates and gel-like states [25][26][27] that could drive compartmentalization of cytologically visible constitutive heterochromatin in interphase nuclei.…”

“…Detailed examination of nucleoli has shown they are not composed of a single condensed phase surrounded by a dilute phase but consist of subcompartments where a secondary condensed phase is contained within the primary condensed phase; the subcompartments have distinct viscosities, surface tensions and protein compositions [66,67]. In vitro studies on mammalian HP1α have shown that HP1α undergoes LLPS and led to the suggestion that HP1α-dependent constitutive heterochromatin might also consist of phase-separated subcompartments; specifically a soluble phase, a liquid droplet phase and a gel-like phase [26,27]. The propensity to form liquid droplets in vitro is peculiar to HP1α because, under the same conditions, HP1β and γ do not form liquid droplets [68]; there seems not to be a relationship between the ability to form liquid droplets and pLI.…”

Immune Regulation in Time and Space: The Role of Local- and Long-Range Genomic Interactions in Regulating Immune Responses

“…Single cell sequencing has convincingly shown that nucleosome positioning is not consistent between cells in a population ( 30 – 32 ). Heterochromatin domains comprising phase-separated droplets are now known to be more dynamic than previously assumed ( 33 , 34 ). Chromatin interactions are similarly variegated: the frequency of DNA loop formation is approximately 30% of alleles at maximum depending on the locus and RNAPII-mediated loops are heterogeneous ( 35 – 37 ).…”

Micellar TIA1 with folded RNA binding domains as a model for reversible stress granule formation

“…Liquid-liquid droplets are typically micrometer, morphologically spherical domains that exhibit liquid-like dynamics in their rapid recovery from photobleaching and solution-state NMR spectra (17). Many liquid droplets or condensates in vitro are metastable and transform over time (16,18) in a process referred to as hardening or…”