Letter | Published:

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin

Nature volume 547, pages 236240 (13 July 2017) | Download Citation


Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands1,2,3. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics4. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.

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We thank R. Cooke for the peltier device, R. Isaac for nucleosome arrays, A. Lyon and P. O’Farrell for discussions, D. Canzio, L. Racki and C. Zhou for helpful comments, P. Schuck for advice on analytical ultracentrifugation, and I. Sterin and R. Almeida for cell culture help. This work was supported by an NSF pre-doctoral fellowship to A.G.L, funding from the UCSF Program for Breakthrough Biomedical Research (PBBR) provided by the Sandler Foundation to S.R., grants from the NIH (8P41GM103481 and 1S10D016229) to A.L.B, and grants from NIH (R01GM108455) and PBBR (New Frontier Research Award) to G.J.N.

Author information


  1. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158, USA

    • Adam G. Larson
    • , Daniel Elnatan
    • , Madeline M. Keenen
    • , David A. Agard
    • , Sy Redding
    •  & Geeta J. Narlikar
  2. Tetrad Graduate Program, University of California, San Francisco, San Francisco, California 94158, USA

    • Adam G. Larson
    • , Daniel Elnatan
    •  & Madeline M. Keenen
  3. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, USA

    • Michael J. Trnka
    • , Jonathan B. Johnston
    •  & Alma L. Burlingame
  4. Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California 94158, USA

    • David A. Agard


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A.G.L developed the overall experimental plan with guidance from G.J.N. and carried out the majority of the experiments and their interpretation. D.E. developed and implemented new software for analysing the SAXS data and helped conceive further experiments, M.J.T. performed and analysed the cross-linking and phosphate mapping mass spectrometry experiments, J.B.J. performed the native mass spectrometry experiments with guidance from A.L.B. D.A.A. provided guidance on SAXS experiments. M.K. performed and analysed the DNA curtains experiments and S.R. oversaw their design and interpretation. G.J.N. and A.G.L. wrote the bulk of the manuscript with major contributions from S.R. G.J.N. oversaw the overall project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Geeta J. Narlikar.

Reviewer Information Nature thanks J. Hansen, E. Selker 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

Excel files

  1. 1.


    This file contains a peptide list from Crosslink Mass Spectrometry analysis.


  1. 1.

    Video 1: 400µM nPhos HP1α transitioning from 4°-25°C

    400µM nPhos HP1α transitioning from 4°-25°C over the course of 2min.

  2. 2.

    Video 2: ΔCTE nPhos HP1α droplets observed in a flow cell at room temperature

    ΔCTE nPhos HP1α droplets observed in a flow cell at room temperature with a 40X air objective in 75mM KCl, 20mM HEPES pH7.2, 1mM DTT.

  3. 3.

    Video 3: Addition of 100uM Sgo peptide to 150µM nPhos HP1α at room temperature

    Addition of 100uM Sgo peptide to 150µM nPhos HP1α at room temperature in 75mM KCl, 20mM HEPES pH7.2, 1mM DTT.

  4. 4.

    Video 4: 15µl of 350µM nPhosHP1 α in 75mM KCl, 20mM HEPES pH7.2 and 1mM DTT pre-mixed and incubated at room temperature for 1 minute

    1000 units of Calf Intestinal Alkaline Phosphatase (NEB) were added(1µL)and mixed. Time-lapse video was taken over approximately 12 minutes using the Hyperlapse application on an iPhone6. The video was sped up 800x in iMovie.

  5. 5.

    Video 5: Cy3 labeled H3K9me3 nucleosomes with nPhos HP1α excited with a red laser and observed at room temperature

    Cy3 labeled H3K9me3 nucleosomes with nPhos HP1α excited with a red laser and observed at room temperature with a 63X oil immersion objective.

  6. 6.

    Video 6: 800µM nPhos HP1α in 75mM KCl, 20mM HEPES pH7.2 and 1mM DTT allowed to form a hydrogel at 22°C for 4 hours

    The interface between the hydrogel and the liquid phase can be seen. Sample was then mixed by pipetting.

  7. 7.

    Video 7: WT HP1α’s (50µM) interaction with DNA visualized in real time

    WT HP1α’s (50µM) interaction with DNA visualized in real time with YOYO-1 labeled DNA curtains.

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