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

Abstract

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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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.

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Acknowledgements

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.

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Authors

Contributions

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.

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The authors declare no competing financial interests.

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

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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

Supplementary

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.

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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). https://doi.org/10.1038/nature22822

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