Letter | Published:

Phase separation drives heterochromatin domain formation

Nature volume 547, pages 241245 (13 July 2017) | Download Citation

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.

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References

  1. 1.

    et al. Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144, 732–744 (2011)

  2. 2.

    & Epigenetic regulation of heterochromatic DNA stability. Curr. Opin. Genet. Dev. 18, 204–211 (2008)

  3. 3.

    & Position-effect variegation, heterochromatin formation, and gene silencing in Drosophila. Cold Spring Harb. Perspect. Biol. 5, a017780 (2013)

  4. 4.

    , & Liquid-liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014)

  5. 5.

    et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012)

  6. 6.

    et al. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123–133 (2015)

  7. 7.

    et al. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. eLife 5, e13571 (2016)

  8. 8.

    et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015)

  9. 9.

    Scheidegger’s rivers, Takayasu’s aggregates and continued fractions. Physica A 170, 463–470 (1991)

  10. 10.

    et al. Reversible liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature (2017)

  11. 11.

    & TALE-light imaging reveals maternally guided, H3K9me2/3-independent emergence of functional heterochromatin in Drosophila embryos. Genes Dev. 30, 579–593 (2016)

  12. 12.

    & Nuclear bodies: the emerging biophysics of nucleoplasmic phases. Curr. Opin. Cell Biol. 34, 23–30 (2015)

  13. 13.

    et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014)

  14. 14.

    et al. Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438, 1116–1122 (2005)

  15. 15.

    et al. Entropy gives rise to topologically associating domains. Nucleic Acids Res. 44, 5540–5549 (2016)

  16. 16.

    & The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. EMBO J. 21, 2664–2671 (2002)

  17. 17.

    , & Spatiotemporal regulation of Heterochromatin Protein 1-alpha oligomerization and dynamics in live cells. Sci. Rep. 5, 12001 (2015)

  18. 18.

    et al. Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev. 21, 2300–2311 (2007)

  19. 19.

    et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012)

  20. 20.

    et al. Coexisting liquid phases underlie nucleolar subcompartments. Cell 165, 1686–1697 (2016)

  21. 21.

    & (eds) New Models of the Cell Nucleus: Crowding, Entropic Forces, Phase Separation, and Fractals. Vol. 307 (Academic Press, 2013)

  22. 22.

    , , & Mapping the number of molecules and brightness in the laser scanning microscope. Biophys. J. 94, 2320–2332 (2008)

  23. 23.

    , , & Nucleation by rRNA dictates the precision of nucleolus assembly. Curr. Biol. 26, 277–285 (2016)

  24. 24.

    , , & Raster image correlation spectroscopy in live cells. Nat. Protocols 5, 1761–1774 (2010)

  25. 25.

    , , & HP1 controls genomic targeting of four novel heterochromatin proteins in Drosophila. EMBO J. 26, 741–751 (2007)

  26. 26.

    et al. Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin. EMBO J. 28, 3785–3798 (2009)

  27. 27.

    Can visco-elastic phase separation, macromolecular crowding and colloidal physics explain nuclear organisation? Theor. Biol. Med. Model. 4, 15 (2007)

  28. 28.

    , & Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proc. Natl Acad. Sci. USA 108, 4334–4339 (2011)

  29. 29.

    et al. Perturbation of nuclear architecture by long-distance chromosome interactions. Cell 85, 745–759 (1996)

  30. 30.

    , , , & Duplication and maintenance of heterochromatin domains. J. Cell Biol. 147, 1153–1166 (1999)

  31. 31.

    et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012)

  32. 32.

    , , & Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method. Biophys. J. 96, 707–716 (2009)

  33. 33.

    et al. FlyBase at 25: looking to the future. Nucleic Acids Res. 45, D663–D671 (2017)

  34. 34.

    et al. Sequence complexity of disordered protein. Proteins 42, 38–48 (2001)

  35. 35.

    Non-globular domains in protein sequences: automated segmentation using complexity measures. Comput. Chem. 18, 269–285 (1994)

  36. 36.

    et al. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 31, 3784–3788 (2003)

  37. 37.

    & A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132 (1982)

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Acknowledgements

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.

Author information

Affiliations

  1. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    • Amy R. Strom
    •  & Gary H. Karpen
  2. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA

    • Amy R. Strom
    • , Mustafa Mir
    • , Xavier Darzacq
    •  & Gary H. Karpen
  3. Albert Einstein College of Medicine, Department of Cell Biology, New York, New York, USA

    • Alexander V. Emelyanov
    •  & Dmitry V. Fyodorov

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Contributions

A.R.S. and G.H.K. conceived the experiments. A.R.S. performed Drosophila, cell culture, imaging, FRAP, hexanediol, and droplet-formation experiments and analysis. M.M. and X.D. performed and analysed the lattice light sheet microscopy. A.V.E. and D.V.F. contributed purified HP1α, and performed glycerol gradient experiments. A.R.S. and G.H.K. wrote the manuscript, and all authors contributed ideas and reviewed the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Gary H. Karpen.

Reviewer Information Nature thanks E. Selker, K. Rippe and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

Videos

  1. 1.

    Video 1: HP1a-GFP embryo imaged on a lattice light sheet microscope over nuclear cycles 13-14

    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

  2. 2.

    Video 2: An xy view of one nucleus over cycle 13

    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

  3. 3.

    Video 3: An xz view of the same nucleus in Video S2 over cycle 13

    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

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DOI

https://doi.org/10.1038/nature22989

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