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Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression

Nature Cell Biology volume 21, pages 1578–1589 (2019)Cite this article

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Abstract

Yes-associated protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a select set of enhancers for target gene activation. How YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized the YAP transcription factor TEAD1 and other YAP-related co-activators, including TAZ, and subsequently induced the transcription of YAP-specific proliferation genes. Super-resolution imaging using assay for transposase-accessible chromatin with photoactivated localization microscopy revealed that the YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA polymerase II, the accessible chromatin domains later acquired RNA polymerase II, transcribing RNA. The removal of the intrinsically-disordered YAP transcription activation domain prevented the formation of YAP condensates and diminished downstream YAP signalling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid–liquid phase separation.

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Fig. 1: YAP has an intrinsic ability to phase separate.
Fig. 2: YAP undergoes phase separation under hyperosmotic stress.
Fig. 3: Endogenous YAP form condensates.
Fig. 4: Cytoplasmic EGFP–YAP selectively enriches different proteins.
Fig. 5: Nuclear EGFP–YAP selectively enriches transcription-related proteins.
Fig. 6: YAP condensates induced by sorbitol treatment alter the 3D organization of accessible chromatin.
Fig. 7: EGFP–YAP nuclear condensate localization with RNAPII during sorbitol treatment.
Fig. 8: Phase separation of YAP is important for its nuclear localization and signalling.

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Data availability

Source data for Figs. 2–8 and Extended Data Figs. 1–5 and 7 are available online. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

The software for identifying, localizing and plotting single-molecule data is freely available after execution of a research license with HHMI. The software for the g(r) and DBscan analysis is freely available from https://github.com/ammondongp/3D_ATAC_PALM.

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Acknowledgements

We thank the members of the J.L.-S. lab for their helpful discussions and critical comments. Support for this work was received from the Howard Hughes Medical Institute (to J.L.-S. and Z.L.), Damon Runyon Cancer Research Foundation (grant no. DRG-2233-15 to D.C.), and intramural research funding from the NIH. We also appreciate the help we received from the Flow Cytometry Core Facility of the NEI/NIH.

Author information

Authors and Affiliations

  1. Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

    Danfeng Cai

  2. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Danfeng Cai, Daniel Feliciano, Peng Dong, Zhe Liu & Jennifer Lippincott-Schwartz

  3. Thoracic and Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Daniel Feliciano & Natalie Porat-Shliom

  4. Department of Chemistry and Chemical Biology, University of California, Merced, CA, USA

    Eduardo Flores & Shahar Sukenik

  5. Department of Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL, USA

    Martin Gruebele & Shahar Sukenik

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Contributions

D.C. and J.L.-S. conceived and designed the study. D.C., D.F., P.D., E.F. and S.S. performed the experiments. D.C. and J.L.-S. wrote the manuscript with constructive input from all authors. M.G., N.P.-S., S.S. and Z.L. provided supervision.

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Correspondence to Jennifer Lippincott-Schwartz.

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Extended data

Extended Data Fig. 1 More characterizations of recombinant YAP protein in vitro.

a, Turbidity measurements of purified EGFP–YAP at different salt, BSA and RNA concentrations. The experiment has been repeated 3 times independently with similar results. b, In vitro turbidity assay showing purified EGFP–YAP phase separates at much lower concentrations than EGFP–YAPΔTAD in the presence of 10% PEG2000. Error bars are SD from three repeats. Centre of the data is mean. n=3 biologically independent experiments. Statistics source data are provided in Source Data Extended Data Fig. 1. Source data.

Source data

Extended Data Fig. 2 EGFP–YAP condensates form in hyperosmotic stress.

a, Imaris 3-D rendering of an EGFP–YAP condensate, and quantification of sphericity of those condensates using Imaris. Centre of the data is mean. Error bars are SD. Scale bar: 0.5µm. (b–d) Live-cell imaging of EGFP–YAP in HEK293T cells showing nuclear and cytoplasmic condensates are able to form with different isoforms of YAP b, and in different hyperosmotic agents c,. d, Live-cell imaging showing different cell types are able to form EGFP–YAP condensates under hyperosmotic stress. Scale bars are 10µm. All the experiments are repeated 3 times independently with similar results. e, Quantification of normalized HEK293T nuclear, cytosolic and total volume before and after sorbitol treatment, and after wash. Centre of the data is mean. Error bars are s.e.m. Two-sided paired t-test. Comparing to volume before treatment. Error bars show s.e.m. n=28 biologically independent samples. (f, g) Representative ratiometric images f, and quantification g, of crowding sensor FRET expressed in the same HEK293T cell before and after 0.2M sorbitol treatment. Rainbow RGB look-up table showing changes in FRET indices. Colour bar: FRET index (a.u.). Two-sided paired t-test. Centre of the data is mean. Error bars show s.e.m. Scale bars are 5µm. n=31 biologically independent samples. Statistics source data are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Endogenous YAP forms condensates.

(a, b) Immunoblotting experiments a, and quantifications of immunofluorescence YAP signal b, indicate that YAP signal is effectively knocked down by YAP siRNA. Two-sided unpaired t-test is used in b. Centre of the data is mean. Error bars show s.e.m. All the experiments are repeated 2 times independently with similar results. n=20 biologically independent samples. c, Schematics of construction of CRISPR knock-in YAP–HaloTag U-2 OS cell line. Live-cell imaging d, and quantification e, showing nuclear YAP–HaloTag condensate labelled by JF549 Halo dye increases in number after PEG 300 treatment. Two-sided unpaired t-test analysis. Centre of the data is mean. Error bars show s.e.m. Scale bars are 10µm. n=213 biologically independent control samples. n=253 biologically independent PEG-treated samples. Statistics source data are provided in Source Data Extended Data Fig. 3. Unprocessed blots are provided in Unprocessed Blots Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Characterization of cytoplasmic YAP condensates.

a, Live-cell imaging showing no colocalization of cytoplasmic EGFP–YAP condensates with P-body component mCherry–GW182 after 0.2 M sorbitol treatment for 20 s in HEK293T cells. b, Magnification of boxed region in (a) and line scan. (c, d) Similar to (a, b), showing no colocalization of EGFP–YAP cytoplasmic condensates with mCherry–Ago2 after 0.2 M sorbitol treatment for 20 s. Scale bars are 10µm in whole-cell view, and are 2µm in magnified view. All the experiments are repeated 3 times independently with similar results. Numerical source data are provided in Source Data Extended Data Fig. 4.

Source data

Extended Data Fig. 5 Characterization of nuclear YAP condensates.

a, Representative immunofluorescence images showing EGFP–YAP nuclear condensates colocalize with endogenous TEAD1 under hyperosmotic stress in U-2 OS cells. b, Magnification of boxed region in a, and line scan. Scale bar: 1µm. c, Live-cell imaging showing mCherry–YAP localizes to EGFP–TAZ condensates and new condensates after hyperosmotic stress in U-2 OS cells. Scale bars in (a) and (c) are 5 µm. All the experiments are repeated 3 times independently with similar results. Numerical source data are provided in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 Dynamic localization of RNAPII relative to EGFP–YAP nuclear condensates after hyperosmotic shock.

a, More examples showing no colocalization of RNAPII (pSer2) with EGFP–YAP condensates at 10min in 0.2M sorbitol. Experiments are same as those in Fig. 7b, EGFP–YAP condensates are auto-thresholded and turned into mask using ImageJ, and pseudo-coloured magenta. RNAPII immunofluorescence is autothresholded and turned into mask using ImageJ, and pseudo-coloured cyan. b, Similar to a, but showing more examples of enhanced localization of RNA Pol ll (pSer2) to the periphery of EGFP–YAP condensates at 2 h in sorbitol. All the experiments are repeated 3 times independently with similar results. Scale bars are 1µm.

Extended Data Fig. 7 EGFP–YAPΔTAD mutant serves as a dominant negative protein that decreases endogenous YAP foci.

a, Immunofluorescence images of YAP–HaloTag U-2 OS cells 10 min in sorbitol treatment, with EGFP–YAPΔTAD overexpression (top row) or without (bottom row). Scale bars are 10µm. b, Quantification showing YAP–HaloTag U-2 OS cells have lower number of YAP–HaloTag endogenous YAP condensates after sorbitol treatment for 10 min, if they overexpress EGFP–YAPΔTAD construct. Two-sided unpaired t-test. Centre of the data is mean. Error bars show s.e.m. n=23 biologically independent EGFP–YAPΔTAD-expressing samples. n=24 biologically independent EGFP–YAPΔTAD-negative samples. Statistics source data are provided in Source Data Extended Data Fig. 8.

Source data

Supplementary information

Supplementary Information

Supplementary Equations for processing raw ATAC-PALM images. gBlock sequence of Yap homology arms with HaloTag-coding sequence.

Reporting Summary

Supplementary Video 1

Video taken on a Zeiss LSM880 confocal scope showing some purified EGFP–YAP condensates wetting the coverslip (stuck to the bottom of the coverslip). The experiment was repeated three times independently with similar results.

Supplementary Video 2

Zeiss LSM880 Airyscan video showing fusion of EGFP–YAP condensates (green) formed in a HEK293T cell after 0.2 M sorbitol treatment. Video starts 1 min after sorbitol treatment. The experiment was repeated three times independently with similar results.

Supplementary Video 3

Zoomed-in video of nuclear condensates in Supplementary Video 2 showing fusion among nuclear EGFP–YAP condensates. Fusion events can be seen at 80–90 s, 129–132 s and 150–156 s. The experiment was repeated three times independently with similar results.

Supplementary Video 4

Zoomed-in video of cytoplasmic condensates in Supplementary Video 2 showing fusion among cytoplasmic EGFP–YAP condensates. Fusion events can be seen at 0–8 s, 16–21 s, 73–100 s and 145–158 s. The experiment was repeated three times independently with similar results.

Supplementary Video 5

3D ATAC-PALM image of the entire nucleus in a control-treated HEK293T cell expressing EGFP–YAP. Accessible chromatin regions labelled by ATAC are mainly diffusive with a few visible small clusters. Imaris software (Bitplane) was used to produce this 3D visualization of the nucleus. The experiment was repeated three times independently with similar results.

Supplementary Video 6

3D ATAC-PALM image of the entire nucleus in 5-min-sorbitol-treated HEK293T cell expressing EGFP–YAP. Huge clusters of accessible chromatins labelled by ATAC are seen. Imaris software (Bitplane) was used to produce this 3D visualization of the nucleus. The experiment was repeated three times independently with similar results.

Supplementary Video 7

Colocalization of accessible chromatin clusters labelled by ATAC (red) and EGFP–YAP nuclear condensates (green) in an HEK293T cell at 5 min of sorbitol treatment. To visualize accessible chromatin clusters, ATAC-PALM localizations were binned within a cubic of 100 nm with a 3D Gaussian filter and a convolution kernel of 3×3×3. Imaris software (Bitplane) was used to produce this 3D visualization of the two channels. The experiment was repeated three times independently with similar results.

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Cai, D., Feliciano, D., Dong, P. et al. Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression. Nat Cell Biol 21, 1578–1589 (2019). https://doi.org/10.1038/s41556-019-0433-z

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