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ISWI catalyzes nucleosome sliding in condensed nucleosome arrays

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI’s mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI’s diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a ‘monkey-bar’ model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

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Fig. 1: Intramolecular chromatin fiber folding does not impede remodeling.
Fig. 2: ISWI partitions into condensates and slides nucleosomes under condensate-forming conditions.
Fig. 3: Nucleosome sliding in condensates visualized by FLIM–FRET.
Fig. 4: ATP hydrolysis powers mobility of ISWI in condensates.
Fig. 5: ATP hydrolysis prevents ISWI-mediated hardening of condensates.
Fig. 6: A simple model qualitatively explains experimental observations.

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Data availability

Large data files that exceed the size limitations or data that underlie more peripheral parts of the study are available upon request from the corresponding authors. Source data are provided with this paper.

Code availability

Code is available from GitHub at https://github.com/StiglerLab/Vizjak_2023 (ref. 86).

References

  1. Burak, Y., Ariel, G. & Andelman, D. Onset of DNA aggregation in presence of monovalent and multivalent counterions. Biophys. J. 85, 2100–2110 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Post, C. B. & Zimm, B. H. Theory of DNA condensation: collapse versus aggregation. Biopolymers 21, 2123–2137 (1982).

    Article  CAS  PubMed  Google Scholar 

  3. Woodcock, C. L. F. Ultrastructure of inactive chromatin. J. Cell Biol. 59, A368 (1973).

    Google Scholar 

  4. Olins, A. L. & Olins, D. E. Spheroid chromatin units (v bodies). Science 183, 330–332 (1974).

    Article  CAS  PubMed  Google Scholar 

  5. Finch, J. T. & Klug, A. Solenoidal model for superstructure in chromatin. Proc. Natl Acad. Sci. USA 73, 1897 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ou, H. D. et al. ChromEMT: visualizing 3D chromatin structure and compaction in interphase and mitotic cells. Science 357, eaag0025 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Maeshima, K. et al. Nucleosomal arrays self-assemble into supramolecular globular structures lacking 30-nm fibers. EMBO J. 35, 1115–1132 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Adhireksan, Z., Sharma, D., Lee, P. L. & Davey, C. A. Near-atomic resolution structures of interdigitated nucleosome fibres. Nat. Commun. 11, 4747 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hsieh, T. H. S. et al. Mapping nucleosome resolution chromosome folding in yeast by Micro-C. Cell 162, 108 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ricci, M. A., Manzo, C., García-Parajo, M. F., Lakadamyali, M. & Cosma, M. P. Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo. Cell 160, 1145–1158 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Gibson, B. A. et al. Organization of chromatin by intrinsic and regulated phase separation. Cell 179, 470–484.e21 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Strickfaden, H. et al. Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells. Cell 183, 1772–1784.e13 (2020).

    Article  CAS  PubMed  Google Scholar 

  13. Zhang, Y., Narlikar, G. J. & Kutateladze, T. G. Enzymatic reactions inside biological condensates. J. Mol. Biol. 433, 166624 (2021).

    Article  CAS  PubMed  Google Scholar 

  14. Hihara, S. et al. Local nucleosome dynamics facilitate chromatin accessibility in living mammalian cells. Cell Rep. 2, 1645–1656 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Kornberg, R. D. & Lorch, Y. Primary role of the nucleosome. Mol. Cell 79, 371–375 (2020).

    Article  CAS  PubMed  Google Scholar 

  16. Kim, J. M. et al. Single-molecule imaging of chromatin remodelers reveals role of ATPase in promoting fast kinetics of target search and dissociation from chromatin. eLife 10, e69387 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Corona, D. F. V. et al. ISWI is an ATP-dependent nucleosome remodeling factor. Mol. Cell 3, 239–245 (1999).

    Article  CAS  PubMed  Google Scholar 

  18. Hamiche, A., Sandaltzopoulos, R., Gdula, D. A. & Wu, C. ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF. Cell 97, 833–842 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Ludwigsen, J., Hepp, N., Klinker, H., Pfennig, S. & Mueller-Planitz, F. Remodeling and repositioning of nucleosomes in nucleosomal arrays. Methods Mol. Biol. 1805, 349–370 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Mueller-Planitz, F., Klinker, H., Ludwigsen, J. & Becker, P. B. The ATPase domain of ISWI is an autonomous nucleosome remodeling machine. Nat. Struct. Mol. Biol. 20, 82–89 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Schram, R. D., Klinker, H., Becker, P. B. & Schiessel, H. Computational study of remodeling in a nucleosomal array. Eur. Phys. J. E 38, 85 (2015).

    Article  PubMed  Google Scholar 

  22. Klinker, H. et al. ISWI remodelling of physiological chromatin fibres acetylated at lysine 16 of histone H4. PLoS ONE 9, e88411 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Boyer, L. A. et al. Functional delineation of three groups of the ATP-dependent family of chromatin remodeling enzymes. J. Biol. Chem. 275, 18864–18870 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Logie, C., Tse, C., Hansen, J. C. & Peterson, C. L. The core histone N-terminal domains are required for multiple rounds of catalytic chromatin remodeling by the SWI/SNF and RSC complexes. Biochemistry 38, 2514–2522 (1999).

    Article  CAS  PubMed  Google Scholar 

  25. Peeples, W. & Rosen, M. K. Mechanistic dissection of increased enzymatic rate in a phase-separated compartment. Nat. Chem. Biol. 17, 693–702 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Poirier, M. G., Bussiek, M., Langowski, J. & Widom, J. Spontaneous access to DNA target sites in folded chromatin fibers. J. Mol. Biol. 379, 772–786 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Poirier, M. G., Oh, E., Tims, H. S. & Widom, J. Dynamics and function of compact nucleosome arrays. Nat. Struct. Mol. Biol. 16, 938–944 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hagerman, T. A. et al. Chromatin stability at low concentration depends on histone octamer saturation levels. Biophys. J. 96, 1944–1951 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gibson, B. A. et al. In diverse conditions, intrinsic chromatin condensates have liquid-like material properties. Proc. Natl Acad. Sci. USA 120, e2218085120 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Goins, A. B., Sanabria, H. & Waxham, M. N. Macromolecular crowding and size effects on probe microviscosity. Biophys. J. 95, 5362–5373 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yang, J. G. & Narlikar, G. J. FRET-based methods to study ATP-dependent changes in chromatin structure. Methods 41, 291–295 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Zhang, M. et al. Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography. Mol. Cell 82, 3000 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Weidemann, T. et al. Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. J. Mol. Biol. 334, 229–240 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Leonard, J. D. & Narlikar, G. J. A nucleotide-driven switch regulates flanking DNA length sensing by a dimeric chromatin remodeler. Mol. Cell 57, 850–859 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Larson, A. G. & Narlikar, G. J. The role of phase separation in heterochromatin formation, function, and regulation. Biochemistry 57, 2540–2548 (2018).

    Article  CAS  PubMed  Google Scholar 

  36. Grüne, T. et al. Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI. Mol. Cell 12, 449–460 (2003).

    Article  PubMed  Google Scholar 

  37. Bhardwaj, S. K. et al. Dinucleosome specificity and allosteric switch of the ISW1a ATP-dependent chromatin remodeler in transcription regulation. Nat. Commun. 11, 5913 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yamada, K. et al. Structure and mechanism of the chromatin remodelling factor ISW1a. Nature 472, 448–453 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Li, L. et al. Structure of the ISW1a complex bound to the dinucleosome. Nat. Struct. Mol. Biol. 31, 266–274 (2024). https://doi.org/10.1038/s41594-023-01174-6

  40. Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699.e16 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Muzzopappa, F., Hertzog, M. & Erdel, F. DNA length tunes the fluidity of DNA-based condensates. Biophys. J. 120, 1288–1300 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ludwigsen, J., Klinker, H. & Mueller-Planitz, F. No need for a power stroke in ISWI-mediated nucleosome sliding. EMBO Rep. 14, 1092–1097 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Harrer, N. et al. Structural architecture of the nucleosome remodeler ISWI determined from cross-linking, mass spectrometry, SAXS, and modeling. Structure 26, 282–294.e6 (2018).

    Article  CAS  PubMed  Google Scholar 

  44. Rudolph, J., Mahadevan, J., Dyer, P. & Luger, K. Poly(ADP-ribose) polymerase 1 searches DNA via a ‘monkey bar’ mechanism. Elife 7, e37818 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Deindl, S. et al. ISWI remodelers slide nucleosomes with coordinated multi-base-pair entry steps and single-base-pair exit steps. Cell 152, 442–452 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Gamarra, N., Johnson, S. L., Trnka, M. J., Burlingame, A. L. & Narlikar, G. J. The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h. eLife 7, e35322 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Dann, G. P. et al. ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference. Nature 548, 607–611 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Clapier, C. R., Längst, G., Corona, D. F., Becker, P. B. & Nightingale, K. P. Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI. Mol. Cell. Biol. 21, 875–883 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Schalch, T., Duda, S., Sargent, D. F. & Richmond, T. J. X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature 436, 138–141 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Verschure, P. J. et al. Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules. EMBO Rep. 4, 861–866 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Beaudouin, J., Mora-Bermúdez, F., Klee, T., Daigle, N. & Ellenberg, J. Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins. Biophys. J. 90, 1878–1894 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Erdel, F., Baum, M. & Rippe, K. The viscoelastic properties of chromatin and the nucleoplasm revealed by scale-dependent protein mobility. J. Phys. Condens. Matter 27, 064115 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Maeshima, K. et al. A transient rise in free Mg2+ ions released from ATP-Mg hydrolysis contributes to mitotic chromosome condensation. Curr. Biol. 28, 444–451.e6 (2018).

    Article  CAS  PubMed  Google Scholar 

  54. Shimamoto, Y., Tamura, S., Masumoto, H. & Maeshima, K. Nucleosome–nucleosome interactions via histone tails and linker DNA regulate nuclear rigidity. Mol. Biol. Cell 28, 1580–1589 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kroschwald, S. et al. Different material states of Pub1 condensates define distinct modes of stress adaptation and recovery. Cell Rep. 23, 3327–3339 (2018).

    Article  CAS  PubMed  Google Scholar 

  56. Munder, M. C. et al. A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy. eLife 5, e09347 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Erdel, F. & Rippe, K. Formation of chromatin subcompartments by phase separation. Biophys. J. 114, 2262–2270 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Schneider, M. W. G. et al. A mitotic chromatin phase transition prevents perforation by microtubules. Nature 609, 183 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Keizer, V. I. P. et al. Live-cell micromanipulation of a genomic locus reveals interphase chromatin mechanics. Science 377, 489–495 (2022).

    Article  CAS  PubMed  Google Scholar 

  60. Erdel, F. et al. Mouse heterochromatin adopts digital compaction states without showing hallmarks of HP1-driven liquid–liquid phase separation. Mol. Cell 78, 236–249.e7 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Irgen-Gioro, S., Yoshida, S., Walling, V. & Chong, S. Fixation can change the appearance of phase separation in living cells. Elife 11, e79903 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Hansen, J. C., Maeshima, K. & Hendzel, M. J. The solid and liquid states of chromatin. Epigenetics Chromatin 14, 50 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Korber, P. & Becker, P. B. Nucleosome dynamics and epigenetic stability. Essays Biochem 48, 63–74 (2010).

    Article  CAS  PubMed  Google Scholar 

  64. Muzzopappa, F. et al. Detecting and quantifying liquid–liquid phase separation in living cells by model-free calibrated half-bleaching. Nat. Commun. 13, 1–15 (2022).

    Article  Google Scholar 

  65. Whitehouse, I., Rando, O. J., Delrow, J. & Tsukiyama, T. Chromatin remodelling at promoters suppresses antisense transcription. Nature 450, 1031–1035 (2007).

    Article  CAS  PubMed  Google Scholar 

  66. Gelbart, M. E., Bachman, N., Delrow, J., Boeke, J. D. & Tsukiyama, T. Genome-wide identification of Isw2 chromatin-remodeling targets by localization of a catalytically inactive mutant. Genes Dev. 19, 942 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Blosser, T. R., Yang, J. G., Stone, M. D., Narlikar, G. J. & Zhuang, X. Dynamics of nucleosome remodelling by individual ACF complexes. Nature 462, 1022–1027 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Tilly, B. C. et al. In vivo analysis reveals that ATP-hydrolysis couples remodeling to SWI/SNF release from chromatin. eLife 10, e69424 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Erdel, F., Schubert, T., Marth, C., Längst, G. & Rippe, K. Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites. Proc. Natl Acad. Sci. USA 107, 19873–19878 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Oppikofer, M. et al. Expansion of the ISWI chromatin remodeler family with new active complexes. EMBO Rep. 18, 1697–1706 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Clapier, C. R., Verma, N., Parnell, T. J. & Cairns, B. R. Cancer-associated gain-of-function mutations activate a SWI/SNF-family regulatory hub. Mol. Cell 80, 712–725.e5 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Hodges, H. C. et al. Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers. Nat. Struct. Mol. Biol. 25, 61–72 (2018).

    Article  CAS  PubMed  Google Scholar 

  73. Elfring, L. K. et al. Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2. Genetics 148, 251–265 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Li, W. et al. Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis. Nat. Cell Biol. 22, 960–972 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Shi, B. et al. UTX condensation underlies its tumour-suppressive activity. Nature 597, 726–731 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    Article  PubMed  Google Scholar 

  77. Klinker, H., Haas, C., Harrer, N., Becker, P. B. & Mueller-Planitz, F. Rapid purification of recombinant histones. PLoS ONE 9, e104029 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Demeler, B. & Gorbet, G. E. in Analytical Ultracentrifugation (eds Uchiyama, S. et al.) 119–143 (Springer, 2016).

  80. Goins, A. B., Sanabria, H. & Waxham, M. N. Macromolecular crowding and size effects on probe microviscosity. Biophys J. 95, 5362–5373 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Digman, M. A., Caiolfa, V. R., Zamai, M. & Gratton, E. The phasor approach to fluorescence lifetime imaging analysis. Biophys. J. 94, 14–16 (2008).

    Article  Google Scholar 

  82. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  83. MultiStackReg (BioImage Informatics Index, 2022); https://biii.eu/multistackreg

  84. Koulouras, G. et al. EasyFRAP-web: a web-based tool for the analysis of fluorescence recovery after photobleaching data. Nucleic Acids Res. 46, 467–472 (2018).

    Article  Google Scholar 

  85. R Core Team. R: a language and environment for statistical computing. R Foundation for Statistical Computing https://www.R-project.org/ (2022).

  86. Vizjak_2023 (GitHub, 2023); https://github.com/StiglerLab/Vizjak_2023

Download references

Acknowledgements

We thank the following members of the Mueller-Planitz lab: S. Albig, M. Khare and S. Härtel for histone purification, M. A. Shegane for donating nucleosome arrays, and A. Lentz for purifying GFP and cloning ISWI–GFP. We thank M. Muernseer (Nanolive) for collecting holotomography data and the Ökten group (TU Munich) for providing a sfGFP plasmid. P.V. acknowledges support from the IRTG SFB 1064. F.M.-P. acknowledges financial support from the Deutsche Forschungsgemeinschaft (SFB1064 A07, MU3613/3-1 and MU3613/8-1); J.S. from the LMU Center for Nanoscience CeNS, a DFG Emmy Noether grant (STI673/2-1) and an ERC Starting Grant (758124); P.B.B. from SFB1064 A01 and BE1140/6-1; and M.H. by St. Jude Children’s Research Hospital, the American Lebanese Syrian Associated Charities and NIH awards R01GM141694 and R01GM135599. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. Petra Vizjak

    Present address: Early Stage Bioprocess Development, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

  2. Nicola Hepp

    Present address: Department of Clinical Genetics, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark

  3. Alessandro Scacchetti

    Present address: Epigenetics Institute and Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

  4. Joseph Bartho

    Present address: European Molecular Biology Laboratory, Heidelberg, Germany

  5. Michaela Smolle

    Present address: ViraTherapeutics GmbH, Rum, Austria

  6. These authors contributed equally: Johannes Stigler, Felix Mueller-Planitz.

Authors and Affiliations

  1. Institute of Physiological Chemistry, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

    Petra Vizjak, Nicola Hepp & Felix Mueller-Planitz

  2. Department of Molecular Biology, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

    Petra Vizjak, Nicola Hepp, Alessandro Scacchetti & Peter B. Becker

  3. Gene Center, Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany

    Dieter Kamp, Joseph Bartho & Johannes Stigler

  4. Core Facility Bioimaging and Walter-Brendel-Centre of Experimental Medicine, Biomedical Center, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

    Mariano Gonzalez Pisfil

  5. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Mario Halic

  6. Department of Physiological Chemistry, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

    Michaela Smolle

  7. BioPhysics Core Facility, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

    Michaela Smolle

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  1. Petra Vizjak
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Contributions

Conceptualization: F.M.-P., J.S. and P.V. Methodology and formal analysis: P.V., J.S., M.G.P., N.H., A.S., D.K. and M.S. Investigation: P.V., M.G.P., A.S., J.B., J.S., D.K., N.H. and M.S. Writing of original draft: F.M.-P., P.V., M.G.P., D.K., M.S., A.S., J.B. and J.S. Visualization: P.V., J.S., D.K., M.S. and M.G.P. Funding acquisition: F.M.-P., J.S., P.B.B. and M.H. Writing—review and editing: all authors. N.H. and A.S. contributed equally.

Corresponding authors

Correspondence to Johannes Stigler or Felix Mueller-Planitz.

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Nature Structural & Molecular Biology thanks John van Noort and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Catalytic activity of ISWI for varying Mg-concentrations.

a, Quality controls for 25mer nucleosome arrays. Left: agarose gel after magnesium precipitation of assembled arrays and the resolubilized pellet. IN, input; SN, supernatant; P, pellet. Competitor DNA derived from the plasmid backbone (<1 kb) was excluded from P. Middle: Not1 digestion (a Not1 site is present in each linker) liberated mostly mononucleosomes, running around 400 bp, but little 197 bp fragments, confirming saturation of most 601 repeats with octamers. Right: BsiWI digestion (all nucleosomes occlude a BsiWI restriction site) for arrays assembled with different octamer amounts. As 601 sites become saturated, digestion is hindered. Arrays were reconstituted 16 times with similar results. b, Increasing Mg2+-concentrations reduce mononucleosome-stimulated ATP hydrolysis rates. Saturating concentrations of ATP (1 mM) and mononucleosomes were used (1.33 µM). Control experiments with three times lower mononucleosome concentrations gave the analogous results. c, Nucleosome array as in Fig. 1a but with different orientation of restriction sites such that the BamHI site is now more peripheral. The array was cut out from the plasmid with HincII (light green) and EcoRI (magenta). d, BamHI accessibility assay as in Fig. 1f but for the array shown in c. e, Left: quantification of gel in d and exponential fits of time courses. Right: rate coefficients from single exponential fits. Bars in b and e are mean values of two independent experiments (dots).

Source data

Extended Data Fig. 2 ISWI-GFP partitions into condensates.

a, GST-labeled GFP does not partition into chromatin condensates. The colocalization experiment was performed with 40 nM of unlabeled 25mer, 10 nM of 25mer-Cy3 and 1.125 µM of GFP-GST. N = 2, with similar results. b, ISWI-GFP concentrations were determined inside condensates and, after centrifugation, in the surrounding solution (bottom) from fluorescence intensities using calibration curves with ISWI-GFP dilutions (top). Two different microscope settings were used to image lower and higher dilutions. Means and SD of two independent replicates are shown. c, Nucleosome sliding time courses as in Fig. 2d. Conditions were identical except that reactions were started by addition of Mg-ATP, not by enzyme. N = 1.

Source data

Extended Data Fig. 3 FLIM-FRET controls.

a, Enrichment of labeled 0N60 mononucleosomes in chromatin condensates. N = 3 biological replicates; bars are averages. The mononucleosome concentration in solution was determined from z-plane above the condensates. b, Whole condensate FRAP of FRET-0N60 nucleosomes to assess their exchange between condensate and solution. Line is an average and shadow SD of eight bleached condensates. One of two independent replicates with similar results is shown. c, Phasor representation of data in Fig. 3b. Upon introduction of the acceptor, the donor’s lifetime distribution moves away from the universal circle line (single exponential lifetimes), consistent with at least two donor populations in different FRET states. The phasor representation makes no assumptions on the number of decay rates nor on specific decay model 75. d, Acceptor bleaching enhances donor fluorescence and lifetime, indicative of FRET. Imaging of FRET-0N60 nucleosomes in chromatin condensates. The acceptor fluorophore was bleached in the left half of the field of view, leading to an increase in donor fluorescence and donor lifetime (right most panel). N = 1. e, FLIM-FRET measurements as in Fig. 3d, but with 5 mM Mg-ATP. Bars are averages of three independent experiments (dots).

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Extended Data Fig. 4 ISWI and nucleosome array mobility inside condensates.

a, Intra-condensate mobility of ISWI-GFP measured by partial-condensate FRAP. Half of a condensate was bleached in presence and absence of Mg-ATP (1 mM). Line is an average and shadow SD of 15 bleached condensates for each condition. N = 4 biological replicates. b, Partial-condensate FRAP of ISWI-GFP (500 nM) in presence of indicated nucleotides (all 0.77 mM), 37 nM 25mer and 1.75 nM Cy3-25mer. c, chromatin and ISWI concentrations as in Fig. 4b, but without Cy3-25mer (80 nM unlabeled 25mer). No nucleotide (8 independent condensates) and ADP-data (7 condensates) were collected in the absence of ATP regenerating system, ATP data in its presence (5 independent condensates). d, ISWI-GFP is more mobile than chromatin in condensates. Dual FRAP of ISWI-GFP and Cy3-labeled condensates formed by 25mer arrays in presence of Mg-ATP (1 mM) and ISWI (100 nM). Four independent replicates show similar results.

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Extended Data Fig. 5 ISWI and Mg2+ affect biophysical properties of condensates.

a, Optical tweezer force readout during condensate fusion. The fusion velocity was determined as the slope of the normalized force data at the inflection point. Incurred forces during fusion were on the order of 1 pN (see Methods). b, Fraction of successful fusion events in 1 mM MgCl2 (orange) and 5 mM MgCl2 (red). Nucleosome concentration: 1170 nM. c, Fusion velocities of condensates measured by optical tweezers containing indicated ISWI concentrations. Data was obtained from the indicated number of independent condensates measured in one experiment. d, ISWI increased viscosity of condensates as reported by enhanced fluorescence of the molecular rotor thioflavin T (ThT). In a high viscosity medium, rotation around the C-C bond (green arrow) is constrained, and the excitation energy is released as fluorescence. Bars: average ThT fluorescence intensities relative to outside medium; error: SD. Six condensates were analyzed for 0 nM and 293 nM ISWI, five for 1170 nM ISWI red circles). One of two independent replicates with similar results is shown. e, Nucleotides (1 mM) and 2 mM instead of 1 mM free Mg2+ show only modest effects on fusion velocities compared to ISWI-AMPPNP. Data were obtained from the indicated number of independent condensates derived from two experiments conducted on different days. Statistical significance was determined by two-sided t-test (p-values: UTP: 3.5e-4, ADP: 0.053, AMPPNP: 4.6e-4, 2 mM MgCl2: 1.4e-12, AMPPNP + ISWI: 2.3e-14). Box plots in c and e show medians and the 25th/75th percentiles, whiskers the 9th and 91st percentiles; asterisks indicate significance levels (n.s.: p > 0.05, *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 1e-4).

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Extended Data Fig. 6 Mechanistic details of the simulated monkey bar mechanism.

a, A model for the independent switching of the strengths of the two nucleosome interaction sites during ISWI´s ATPase cycle. Escape rates (black) and transition rates (red) in timestep-1 are indicated. b, Schematic representation of the implementation of the model in Fig. 6a for molecular dynamics simulations. c, Modified Lennard-Jones potential used in simulations. Below distances of 2R0 a regular Lennard-Jones potential is used. Between 2R0 and 3R0 the potential is described using a linear approximation, while interactions with range above 3R0 are set to 0. d, Conversion of the strength of the modified Lennard-Jones potential to escape rates based on the mean first passage time of potential escape76.

Supplementary information

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Supplementary Table 1.

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Supplementary Video 1

FLIM–FRET time lapse with ATP.

Supplementary Video 2

FLIM–FRET time lapse with AMPPNP.

Supplementary Video 3

Controlled fusion with optical tweezers of chromatin condensates containing ISWI and ADP–BeFx.

Supplementary Video 4

Controlled fusion with optical tweezers of chromatin condensates containing ISWI and ATP.

Supplementary Video 5

Simulation of ISWI FRAP with graph.

Supplementary Video 6

Simulation of condensate fusion with graph.

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Vizjak, P., Kamp, D., Hepp, N. et al. ISWI catalyzes nucleosome sliding in condensed nucleosome arrays. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01290-x

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