Phase separation drives heterochromatin domain formation

Abstract

Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability1, and silencing of transposon and gene expression2. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA3. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments4. We show that Drosophila HP1a protein undergoes liquid–liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. We conclude that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. We propose that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.

De Novo Prediction of Human Chromosome Structures: Epigenetic Marking Patterns Encode Genome Architecture

“…The accuracy achieved by using our energy landscape model in predicting the effects of compartmentalization, as seen by Hi-C and 3D FISH, supports the plausibility of microphase separation being the physical process driving compartmentalization in chromosomes (17,(34)(35)(36) (Fig. 4).…”

Genomic and Proteomic Resolution of Heterochromatin and Its Restriction of Alternate Fate Genes

“…These findings may explain activation of repeat elements and the altered organization of compacted chromatin in ALS models (Amlie-Wolf et al, 2015; Li et al, 2012). TDP-43 and FUS mediate liquid-liquid phase separation in stress granules via their low-complexity domains (Taylor et al, 2016), raising the possibility that they may perform similar functions at heterochromatin domains, which also form via phase separation in an HP1-dependent manner (Larson et al, 2017; Strom et al, 2017). We found that 20 of 172 H3K9me3 heterochromatin proteins contain FUS-like RGG domains, as curated by (Thandapani et al, 2013), versus 1 in 1,474 Gradient Top proteins (Table S5), indicating that a motif capable of mediating phase separation is enriched among H3K9me3 heterochromatin proteins.…”

Disruption of an RNA-binding hinge region abolishes LHP1-mediated epigenetic repression

“…Such regions can undergo phase transitions to form non-membranebound subcellular compartments, frequently containing RNA (Lin et al 2015). It is interesting to speculate that the subnuclear foci that we observed may therefore represent a phase-separated compartment, as observed recently for mammalian HP1α (Larson et al 2017) and Drosophila HP1a (Strom et al 2017). RNA-binding to the LHP1 hinge region could precipitate formation of nuclear LHP1 bodies to maintain a locally high concentration of LHP1 at Polycomb target genes.…”