Article | Published:

Acetylation of intrinsically disordered regions regulates phase separation

Abstract

Liquid–liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

Published research reagents from the FMI are shared with the academic community under a Material Transfer Agreement (MTA) having terms and conditions corresponding to those of the UBMTA (Uniform Biological Material Transfer Agreement).

Additional information

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

References

  1. 1.

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

  2. 2.

    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).

  3. 3.

    Tompa, P. Intrinsically disordered proteins: a 10-year recap. Trends Biochem. Sci. 37, 509–516 (2012).

  4. 4.

    Protter, D. S. W. & Parker, R. Principles and properties of stress granules. Trends Cell Biol. 26, 668–679 (2016).

  5. 5.

    Tyedmers, J., Mogk, A. & Bukau, B. Cellular strategies for controlling protein aggregation. Nat. Rev. Mol. Cell Biol. 11, 777–788 (2010).

  6. 6.

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

  7. 7.

    Patel, A. et al. A liquid-to-solid phase transition of the ALS Protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015).

  8. 8.

    Brangwynne, C. P., Tompa, P. & Pappu, R. V. Polymer physics of intracellular phase transitions. Nat. Phys. 11, 899–904 (2015).

  9. 9.

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

  10. 10.

    Hofweber, M. et al. Phase separation of FUS Is suppressed by its nuclear import receptor and arginine methylation. Cell 173, 706–719 e713 (2018).

  11. 11.

    Monahan, Z. et al. Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. EMBO J. 36, 2951–2967 (2017).

  12. 12.

    Kawaguchi, Y. et al. The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115, 727–738 (2003).

  13. 13.

    Kwon, S., Zhang, Y. & Matthias, P. The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response. Genes Dev. 21, 3381–3394 (2007).

  14. 14.

    Zhang, Y. et al. HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. EMBO J. 22, 1168–1179 (2003).

  15. 15.

    Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458 (2002).

  16. 16.

    Matthias, P., Yoshida, M. & Khochbin, S. HDAC6 a new cellular stress surveillance factor. Cell Cycle 7, 7–10 (2008).

  17. 17.

    Boyault, C., Sadoul, K., Pabion, M. & Khochbin, S. HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination. Oncogene 26, 5468–5476 (2007).

  18. 18.

    Tourrière, H. et al. The RasGAP-associated endoribonuclease G3BP assembles stress granules. J. Cell Biol. 160, 823–831 (2003).

  19. 19.

    Legros, S. et al. The HTLV-1 Tax protein inhibits formation of stress granules by interacting with histone deacetylase 6. Oncogene 30, 4050–4062 (2011).

  20. 20.

    Schölz, C. et al. Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat. Biotechnol. 33, 415–423 (2015).

  21. 21.

    Bannister, A. J., Miska, E. A., Görlich, D. & Kouzarides, T. Acetylation of importin-alpha nuclear import factors by CBP/p300. Curr. Biol. 10, 467–470 (2000).

  22. 22.

    Zhang, X. et al. HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol. Cell 27, 197–213 (2007).

  23. 23.

    Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 (2009).

  24. 24.

    Zhang, L. et al. Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity. Protein Cell 6, 42–54 (2015).

  25. 25.

    Shih, J. W. et al. Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response. Biochem. J. 441, 119–129 (2012).

  26. 26.

    North, B. J., Marshall, B. L., Borra, M. T., Denu, J. M. & Verdin, E. The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase. Mol. Cell 11, 437–444 (2003).

  27. 27.

    Hao, R. et al. Proteasomes activate aggresome disassembly and clearance by producing unanchored ubiquitin chains. Mol. Cell 51, 819–828 (2013).

  28. 28.

    Grozinger, C. M., Hassig, C. A. & Schreiber, S. L. Three proteins define a class of human histone deacetylases related to yeast Hda1p. Proc. Natl Acad. Sci. USA 96, 4868–4873 (1999).

  29. 29.

    Hai, Y. & Christianson, D. W. Histone deacetylase 6 structure and molecular basis of catalysis and inhibition. Nat. Chem. Biol. 12, 741–747 (2016).

  30. 30.

    Miyake, Y. et al. Structural insights into HDAC6 tubulin deacetylation and its selective inhibition. Nat. Chem. Biol. 12, 748–754 (2016).

  31. 31.

    Thompson, P. R. et al. Regulation of the p300 HAT domain via a novel activation loop. Nat. Struct. Mol. Biol. 11, 308–315 (2004).

  32. 32.

    Floor, S. N., Condon, K. J., Sharma, D., Jankowsky, E. & Doudna, J. A. Autoinhibitory interdomain interactions and subfamily-specific extensions redefine the catalytic core of the human DEAD-box protein DDX3. J. Biol. Chem. 291, 2412–2421 (2016).

  33. 33.

    Wang, A. et al. A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. EMBO J. 37, e97452 (2018).

  34. 34.

    Li, X. et al. The repeat region of cortactin is intrinsically disordered in solution. Sci. Rep. 7, 16696 (2017).

  35. 35.

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

  36. 36.

    Youn, J. Y. et al. High-density proximity mapping reveals the subcellular organization of mrna-associated granules and bodies. Molecular cell 69, 517–532 e511 (2018).

  37. 37.

    Jain, S. et al. ATPase-modulated stress granules contain a diverse proteome and substructure. Cell 164, 487–498 (2016).

  38. 38.

    Protter, D. S. W. et al. Intrinsically disordered regions can contribute promiscuous interactions to RNP granule assembly. Cell Rep. 22, 1401–1412 (2018).

  39. 39.

    Wheeler, J. R., Matheny, T., Jain, S., Abrisch, R. & Parker, R. Distinct stages in stress granule assembly and disassembly. eLife 5, e18413 (2016).

  40. 40.

    Markmiller, S. et al. Context-dependent and disease-specific diversity in protein interactions within stress granules. Cell 172, 590–604 e513 (2018).

  41. 41.

    Riback, J. A. et al. Stress-triggered phase separation is an adaptive, evolutionarily tuned response. Cell 168, 1028–1040.e1019 (2017).

  42. 42.

    Fanfoni, M. & Tomellini, M. The Johnson-Mehl-Avrami-Kohnogorov model: a brief review. IlNuovo Cimento D 20, 1171–1182 (1998).

  43. 43.

    Valentin-Vega, Y. A. et al. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation. Sci. Rep. 6, 25996 (2016).

  44. 44.

    Elbaum-Garfinkle, S. et al. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl Acad. Sci. USA 112, 7189–7194 (2015).

  45. 45.

    Ohn, T., Kedersha, N., Hickman, T., Tisdale, S. & Anderson, P. A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly. Nat. Cell Biol. 10, 1224–1231 (2008).

  46. 46.

    Jedrusik-Bode, M. et al. The sirtuin SIRT6 regulates stress granule formation in C. elegans and mammals. J. Cell Sci. 126, 5166–5177 (2013).

  47. 47.

    Cohen, T. J. et al. An acetylation switch controls TDP-43 function and aggregation propensity. Nat. Commun. 6, 5845 (2015).

  48. 48.

    Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science 361, eaar3958 (2018).

  49. 49.

    Chong, S. et al. Imaging dynamic and selective low-complexity domain interactions that control gene transcription. Science 361, eaar2555 (2018).

  50. 50.

    Hnisz, D., Shrinivas, K., Young, R. A., Chakraborty, A. K. & Sharp, P. A. A phase separation model for transcriptional control. Cell 169, 13–23 (2017).

  51. 51.

    Banerjee, I. et al. Influenza A virus uses the aggresome processing machinery for host cell entry. Science 346, 473–477 (2014).

  52. 52.

    Ostapcuk, V. et al. Activity-dependent neuroprotective protein recruits HP1 and CHD4 to control lineage-specifying genes. Nature 557, 739–743 (2018).

  53. 53.

    Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

  54. 54.

    Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526 (2014).

  55. 55.

    Hubner, N. C. et al. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 189, 739–754 (2010).

  56. 56.

    Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

  57. 57.

    Mittasch, M. et al. Non-invasive perturbations of intracellular flow reveal physical principles of cell organization. Nat. Cell Biol. 20, 344–351 (2018).

Download references

Acknowledgements

We are grateful to T. Hyman for use of the microscope with thermal stage on short notice and for comments on the manuscript, and R. Voit (German Cancer Research Center, Heidelberg) for HAT expression vectors. We thank L. Gelman and S. Bourke for help with microscopic analysis, H. Kohler for FACS analysis, J. Seebacher and V. Iesmantavicius for interpretation of mass spectrometry data, H. Gut for help with structure predictions, M.B. Stadler for acetylome-wide IDR analysis, J. Wilbertz for help with live-cell imaging, L. Giorgetti and Y. Zhan for help with mathematical modeling, W. Filipowicz and J. Chao for critical comments on the manuscript. We thank C. Schölz for valuable suggestions. We also thank L. Wang for advice on protein purification, G. Matthias and C. Cao for their helpful technical assistance, Y. Miyake for providing us with biological materials for experiments, R. Clerc for critical comments on the manuscript, and all the Matthias laboratory members for fruitful discussions. M. Saito is supported in part by a fellowship from the Nakajima Foundation. A.W. Fritsch is supported by the ELBE postdoctoral fellows program. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). This work was supported by the Novartis Research Foundation.

Author information

M.S. and P.M. designed the project; M.S. performed all experiments and interpreted the data for the manuscript under the supervision of P.M.; M.S. and D.H. performed mass spectrometry analysis; M.S. and J.E. performed microscopy and image analysis; M.S., A.W.F. and M.K. performed temperature-dependent microscopy measurements and their image analysis; M.S. and B.T.W. analyzed HDAC6 related acetylome data under the supervision of C.C.; M.S. and P.M. wrote the manuscript and all authors contributed to the final version.

Competing interests

The authors declare no competing interests.

Correspondence to Patrick Matthias.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–27

  2. Reporting Summary

  3. Supplementary Dataset 1

    Total DDX3X SG volume (related to Fig. 5)

  4. Supplementary Dataset 2

    DDX3X-interactome (related to Fig. 6)

  5. Supplementary Dataset 3

    Raw data used for mathematical modeling of SG growth (related to Fig. 6)

  6. Supplementary Video 1

    Fusion behavior of DDX3X-IDR1 droplet (related to Fig. 3)

  7. Supplementary Video 2

    DDX3X-IDR1 droplet disappearance by LLPS following a temperature increase (related to Fig. 3)

  8. Supplementary Video 3

    Liquid-like properties of mCherry-DDX3X SGs (related to Fig. 4)

  9. Supplementary Video 4

    SG formation of WT DDX3X and its mutants (related to Fig. 6)

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

  • Received

  • Accepted

  • Published

  • Issue Date

DOI

https://doi.org/10.1038/s41589-018-0180-7

Further reading

Fig. 1: DDX3X-IDR1 is specifically deacetylated by HDAC6.
Fig. 2: Stress induces acetylation of DDX3X and other proteins.
Fig. 3: Acetylation of DDX3X-IDR1 impairs its droplet formation by LLPS in vitro.
Fig. 4: Acetyl-mimic/dead mutations alter DDX3X SG dynamics.
Fig. 5: Deacetylation of DDX3X is required for normal SG size.
Fig. 6: SG maturation is promoted by deacetylation of DDX3X.