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.
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Funding was provided by National Institute of Health grants R01 GM117420 (G.H.K.), R01-GM074233 (D.V.F.), and U54-DK107980 and U01-EB021236 (X.D.), and the California Institute for Regenerative Medicine (CIRM, LA1-08013, X.D.). We thank A. Dernburg for use of the spinning disk confocal, M. Scott for maintenance of the rastering confocal, S. Colmenares for cell lines, C. Robertson for introduction to RICS and N&B, A. Tangara for help in assembling and maintaining the LLSM, and A. Dernburg, S. Safran, M. Elbaum and the Karpen laboratory for helpful comments on the manuscript.
Extended data figures
HP1a-GFP embryo imaged on a lattice light sheet microscope over nuclear cycles 13-14. Note formation, growth, fusion and maturation of HP1a foci. A higher quality version of this video can be viewed here https://youtu.be/o3NV22f_8Wk
An xy view of one nucleus over cycle 13, imaged on a lattice light sheet microscope. A higher quality version of this video can be viewed here https://youtu.be/tqHV7hQrVME
An xz view of the same nucleus in Video S2 over cycle 13, imaged on a lattice light sheet microscope. A higher quality version of this video can be viewed here https://youtu.be/SUcAOTKXXVs
About this article
Nature Reviews Neurology (2019)