Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Phase separation by HP1α.
Figure 2: NTE Phosphorylation promotes HP1α oligomerization and conformational change.
Figure 3: Consequences of interactions with DNA.
Figure 4: Partitioning of specific macromolecules into HP1α phase and behaviour of HP1α molecules in cells.


  1. Kang, J. et al. Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells. Mol. Biol. Cell 22, 1181–1190 (2011)

    CAS  Article  Google Scholar 

  2. Eissenberg, J. C. & Elgin, S. C. The HP1 protein family: getting a grip on chromatin. Curr. Opin. Genet. Dev. 10, 204–210 (2000)

    CAS  Article  Google Scholar 

  3. Ye, Q., Callebaut, I., Pezhman, A., Courvalin, J. C. & Worman, H. J. Domain-specific interactions of human HP1-type chromodomain proteins and inner nuclear membrane protein LBR. J. Biol. Chem. 272, 14983–14989 (1997)

    CAS  Article  Google Scholar 

  4. Greene, E. C., Wind, S., Fazio, T., Gorman, J. & Visnapuu, M.-L. in Single Molecule Tools: Fluorescence Based Approaches, Part A (ed. Walter, N. G. ) Volume 472, 293–315 (Academic Press, 2010)

    Google Scholar 

  5. Nishibuchi, G. et al. N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity. Nucleic Acids Res. 42, 12498–12511 (2014)

    CAS  Article  Google Scholar 

  6. Canzio, D., Larson, A. & Narlikar, G. J. Mechanisms of functional promiscuity by HP1 proteins. Trends Cell Biol. 24, 377–386 (2014)

    CAS  Article  Google Scholar 

  7. Vakoc, C. R., Mandat, S. A., Olenchock, B. A. & Blobel, G. A. Histone H3 lysine 9 methylation and HP1γ are associated with transcription elongation through mammalian chromatin. Mol. Cell 19, 381–391 (2005)

    CAS  Article  Google Scholar 

  8. Locke, J., Kotarski, M. A. & Tartof, K. D. Dosage-dependent modifiers of position effect variegation in Drosophila and a mass action model that explains their effect. Genetics 120, 181–198 (1988)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Smothers, J. F. & Henikoff, S. The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins. Mol. Cell. Biol. 21, 2555–2569 (2001)

    CAS  Article  Google Scholar 

  10. Kilic, S., Bachmann, A. L., Bryan, L. C. & Fierz, B. Multivalency governs HP1α association dynamics with the silent chromatin state. Nat. Commun. 6, 7313 (2015)

    CAS  ADS  Article  Google Scholar 

  11. Mishima, Y. et al. Hinge and chromoshadow of HP1α participate in recognition of K9 methylated histone H3 in nucleosomes. J. Mol. Biol. 425, 54–70 (2013)

    CAS  Article  Google Scholar 

  12. Hiragami-Hamada, K. et al. N-terminal phosphorylation of HP1α promotes its chromatin binding. Mol. Cell. Biol. 31, 1186–1200 (2011)

    CAS  Article  Google Scholar 

  13. LeRoy, G. et al. Heterochromatin protein 1 is extensively decorated with histone code-like post-translational modifications. Mol. Cell. Proteomics 8, 2432–2442 (2009)

    CAS  Article  Google Scholar 

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

    CAS  ADS  Article  Google Scholar 

  15. Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017)

    CAS  Article  Google Scholar 

  16. Velez, G. et al. Evidence supporting a critical contribution of intrinsically disordered regions to the biochemical behavior of full-length human HP1γ. J. Mol. Model. 22, 12 (2016)

    Article  Google Scholar 

  17. Chipuk, J. E. et al. Direct Activation of Bax by p53mediates mitochondrial membranepermeabilization and apoptosis. Science 303, 1–6 (2004)

    Article  Google Scholar 

  18. Canzio, D. et al. A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assembly. Nature 496, 377–381 (2013)

    CAS  ADS  Article  Google Scholar 

  19. Hiragami-Hamada, K. et al. Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin. Nat. Commun. 7, 11310 (2016)

    CAS  ADS  Article  Google Scholar 

  20. Sugimoto, K. et al. Human homolog of Drosophila heterochromatin-associated protein 1 (HP1) is a DNA-binding protein which possesses a DNA-binding motif with weak similarity to that of human centromere protein C (CENP-C)1. J. Biochem. 120, 153–159 (2005)

    Article  Google Scholar 

  21. Azzaz, A. M. et al. Human heterochromatin protein 1α promotes nucleosome associations that drive chromatin condensation. J. Biol. Chem. 289, 6850–6861 (2014)

    CAS  Article  Google Scholar 

  22. Fuller, D. N. et al. Measurements of single DNA molecule packaging dynamics in bacteriophage lambda reveal high forces, high motor processivity, and capsid transformations. J. Mol. Biol. 373, 1113–1122 (2007)

    CAS  Article  Google Scholar 

  23. Hyman, A. A., Weber, C. A. & Jülicher, F. Liquid-liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014)

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  25. Richter, K., Nessling, M. & Lichter, P. Macromolecular crowding and its potential impact on nuclear function. Biochim. Biophys. Acta 1783, 2100–2107 (2008)

    CAS  Article  Google Scholar 

  26. Strom, A. R. et al. Phase separation drives heterochromatin domain formation. Nature (2017)

  27. Li, C. et al. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnol. 11, 92 (2011)

    CAS  Article  Google Scholar 

  28. Schuck, P. On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation. Anal. Biochem. 320, 104–124 (2003)

    CAS  Article  Google Scholar 

  29. Brautigam, C. A. Calculations and publication-quality illustrations for analytical ultracentrifugation data. Methods Enzymol. 562, 109–133 (2015)

    CAS  Article  Google Scholar 

  30. Hansen, S. Bayesian estimation of hyperparameters for indirect Fourier transformation in small-angle scattering. J. Appl. Crystallogr. 33, 1415–1421 (2000)

    CAS  Article  Google Scholar 

  31. Kaustov, L. et al. Recognition and specificity determinants of the human cbx chromodomains. J. Biol. Chem. 286, 521–529 (2011)

    CAS  Article  Google Scholar 

  32. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protocols 1, 2856–2860 (2006)

    CAS  Article  Google Scholar 

  33. Trnka, M. J., Baker, P. R., Robinson, P. J. J., Burlingame, A. L. & Chalkley, R. J. Matching cross-linked peptide spectra: only as good as the worse identification. Mol. Cell. Proteomics 13, 420–434 (2014)

    CAS  Article  Google Scholar 

  34. Lu, J. et al. Improved peak detection and deconvolution of native electrospray mass spectra from large protein complexes. J. Am. Soc. Mass Spectrom. 26, 2141–2151 (2015)

    CAS  ADS  Article  Google Scholar 

  35. Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015)

    CAS  Article  Google Scholar 

  36. Correll, S. J., Schubert, M. H. & Grigoryev, S. A. Short nucleosome repeats impose rotational modulations on chromatin fibre folding. EMBO J. 31, 2416–2426 (2012)

    CAS  Article  Google Scholar 

  37. Gallardo, I. F. et al. High-throughput universal DNA curtain arrays for single-molecule fluorescence imaging. Langmuir 31, 10310–10317 (2015)

    CAS  Article  Google Scholar 

Download references


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

Authors and Affiliations



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.

Corresponding author

Correspondence to Geeta J. Narlikar.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

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 figures and tables

Extended Data Figure 1 Mass-spectrometric analysis of HP1α proteins.

Cross-linking mass spectrometry of HP1α identifies extensive interactions between the CSD and the hinge region. a, Phosphorylation of HP1α occurs almost exclusively at the N terminus. Left, annotated HCD (higher energy collision dissociation) product ion spectra of a quadruply phosphorylated, doubly charged HP1α peptide at Ser11, Ser12, Ser13, Ser14. Neutral loss of phosphoric acid from b-ions is indicated by b*. Right, relative occupancy of observed HP1α phosphorylation sites as estimated by spectral counting. 41.7% of product ion spectra from peptides containing serine at residues 11–14 were observed quadruply phosphorylated (393 of 943 spectra). An additional 32.9% (310 spectra), 12.8% (121 spectra), and 8.5% (80 spectra) were identified triply, doubly, and singly phosphorylated, respectively, while only 4.1% (39 spectra) were observed with no phosphorylation. By contrast, phosphorylation was observed at other positions (Ser45, Thr132 and/or Ser135, Thr145, and Thr 188) with 1–2.5% occupancy (1,059, 2,243, 1,586, 1,042 total spectra observed for peptides containing these residues). b, Native MS charge state envelopes for wild-type, Phos- and nPhos-HP1α. c, Table with predicted and observed masses is also shown. The deconvoluted masses fit best to dimeric HP1α modified by eight phosphates in Phos-HP1α and nPhos-HP1α samples. d, Cross-links were identified by separating cross-linked HP1α by SDS–PAGE and excising bands corresponding to monomeric and dimeric HP1α. Putative inter-protein cross-links, diagrammed here, were identified by taking the set of cross-links that are unique to the dimer band (from three replicates). Only cross-links identified by four or more product ion spectra are shown for clarity.

Extended Data Figure 2 Phase separation is an isoform-specific capability of phosphorylated HP1α that is perturbed by GFP fusion.

a, 1 μl of a 400 μM solution of each protein was spotted on a plastic coverslip and imaged at 10×. Scale bars, 50 μm. Buffer was 75 mM KCl, 20 mM HEPES pH 7.2, 1 mM DTT. Phos-HP1α is phosphorylated in the N terminus and hinge, nE-HP1α has the N-terminal serines replaced with glutamate, Phos-GFP–HP1α is a N-terminal GFP fusion phosphorylated in the N terminus and hinge, Phos-HP1α–GFP is a C-terminal GFP fusion phosphorylated in the N terminus and hinge, Phos-HP1α(BPM) has the KRK hinge sequence mutated to alanines and is phosphorylated in the N terminus and hinge, Phos-HP1α–KCK has a C-terminal GSKCK tag added and is phosphorylated in the N terminus and hinge. b, Complete comparison of saturation concentration measurements between spin-down assay (left) and 340 nm turbidity-based measurement (right), some data are repeated from Fig. 1.

Extended Data Figure 3 Estimation of oligomeric potential by sedimentation velocity analytical ultracentrifugation.

a, Representative sedimentation velocity runs from high-concentration HP1 samples. Percentage of the loaded sample higher than 6 S was quantified to estimate oligomeric species higher than a dimer. b, Table showing the comparison of high-concentration AUC runs. Average sedimentation coefficient was quantified by integrating from 1–20 S and higher order oligomers were estimated by integrating signal from 6–20 S. c, Analytical ultracentrifugation c(S) analysis of fully phosphorylated HP1α and the fully phosphorylated basic patch mutant. d, Analytical centrifugation c(S) analysis of fully phosphorylated HP1α and the fully phosphorylated HP1α/β chimaera (PhosNH-α/βchimaera). Representative traces from three independent experiments are shown in ad (n = 3).

Extended Data Figure 4 Estimation of HP1α dimerization affinity by isothermal calorimetry and analytical ultracentrifugation.

a, Isothermal calorimetry data showing the measured dimerization Kd for the HP1α CSD. The calculated Kd is 1.1 μM. b, An analytical ultracentrifugation isotherm used to estimate the dimerization Kd for wild-type HP1α. Estimated Kd for dimerization using an isodesmic association model is 1.12 μM.

Extended Data Figure 5 Scattering and Guinier fits of SAXS on wild-type and nPhos-HP1α show homogeneous populations.

a, Raw X-ray scattering intensity of wild-type (blue points) and nPhos-HP1 (green points) at 3.5 mg ml−1 (150 μM) concentration. Black lines are Fourier transforms of the fitted interatomic distance distribution, P(r), with χ2 values of 1.186 and 1.199 for wild type and nPhos, respectively. b, Guinier plots of wild-type (blue points) and nPhos-HP1 (green points) at 150 μM. Black lines are linear fits to the data plotted as log intensity versus q2. The range of data used in the linear fits extend up to q × Rg of 1.3. Rg is radius of gyration and q is scattering vector. The corresponding residuals for each fit are shown below as vertically shifted horizontal lines for clarity.

Extended Data Figure 6 Phosphorylated HP1α elutes as an extended dimer when examined by SEC-MALS.

a, Elution profiles of wild-type HP1 and nPhos-HP1 examined by SEC-MALS. The horizontal green, and blue lines correspond to the calculated masses for nPhos-HP1 and wild-type HP1, respectively. b, MALS trace of fully phosphorylated HP1α run under identical conditions to those in a.

Extended Data Figure 7 Measuring shogushin 1, lamin B receptor, H3K9me3 peptide affinity, and the effect of shogushin peptide binding on oligomerization.

a, b, Fluorescence anisotropy plots showing the Kd measurements (values in μM next to symbols for wild-type versus HP1α(CSDm)) for LBR and Sgo1 peptide binding to wild-type HP1α and the I163A CSD mutant (CSDm), which can no longer form dimers. c, Comparative analytical ultracentrifugation runs of approximately 50 μM nPhos HP1α with and without 100 μM shogushin or LBR. d, Fluorescence anisotropy plots with a 15-mer trimethylated H3K9 peptide showing the relevant HP1 isoforms can bind the nucleosome tail.

Extended Data Figure 8 Effects of additional ligands on saturation concentrations.

a, Bar graphs displaying the effects of 100 μM of the polyamine spermine along with the H3K9 and H3K9me3 peptides on phase-separation behaviour. b, Schematic of the assay used to quantify the partitioning of Cy3-labelled substrates into the two phases. The blue bars represent the total concentration of the labelled species before spin down; the orange bars represent the concentration of Cy3-labelled species remaining in the upper phase after spin down. The lower phase contains HP1α at a higher concentration than in the upper phase. Error bars represent standard error of the mean from three independent measurements. c, Model for phosphorylation or DNA-driven HP1α phase separation. Phosphorylation or DNA binding relieves intra-HP1 contacts and opens up the dimer. The location(s) of the intra- and inter-dimer contacts that change during this transition are not fully understood, but are predicted to involve interactions between the CTE, hinge and NTE.

Extended Data Figure 9 Consequences of the interaction between HP1 and DNA.

a, b, Wide-field TIRF microscopy images of DNA compaction by HP1β (a) and HP1α(BPM) (b) at different time points. Scale bars, 5 μm. c, d, Average kymograms for HP1β (c; n = 368) and HP1α(BPM) (d; n = 318) overlayed with fits for average compaction speed (dashed line) and standard deviation (solid lines). e, f, Individual kymograms showing compaction by wild-type HP1α (e) and nPhos-HP1α (f) at different protein concentrations.

Extended Data Figure 10 Additional micrographs of NIH3T3 cells transduced with HP1.

NIH3T3 cells transduced with 0.3 μg of HP1 proteins and imaged under identical conditions. a, nPhos-HP1α; b, HP1α (CSDm); c, wild-type HP1α.

Supplementary information


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

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

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

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.

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.

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.

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.

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.

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.

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Larson, A., Elnatan, D., Keenen, M. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing