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Single-stranded nucleic acid binding and coacervation by linker histone H1

Nature Structural & Molecular Biology volume 29, pages 463–471 (2022)Cite this article

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Abstract

The H1 linker histone family is the most abundant group of eukaryotic chromatin-binding proteins. However, their contribution to chromosome structure and function remains incompletely understood. Here we use single-molecule fluorescence and force microscopy to directly visualize the behavior of H1 on various nucleic acid and nucleosome substrates. We observe that H1 coalesces around single-stranded DNA generated from tension-induced DNA duplex melting. Using a droplet fusion assay controlled by optical tweezers, we find that single-stranded nucleic acids mediate the formation of gel-like H1 droplets, whereas H1–double-stranded DNA and H1–nucleosome droplets are more liquid-like. Molecular dynamics simulations reveal that multivalent and transient engagement of H1 with unpaired DNA strands drives their enhanced phase separation. Using eGFP-tagged H1, we demonstrate that inducing single-stranded DNA accumulation in cells causes an increase in H1 puncta that are able to fuse. We further show that H1 and Replication Protein A occupy separate nuclear regions, but that H1 colocalizes with the replication factor Proliferating Cell Nuclear Antigen, particularly after DNA damage. Overall, our results provide a refined perspective on the diverse roles of H1 in genome organization and maintenance, and indicate its involvement at stalled replication forks.

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Fig. 1: H1 coalesces around nascent ssDNA.
Fig. 2: H1 exhibits enhanced phase separation with single-stranded nucleic acids.
Fig. 3: H1 droplets exhibit distinct material properties depending on the nucleic acid/chromatin substrate.
Fig. 4: Multivalency and interaction strength influence H1–DNA phase separation.
Fig. 5: H1 puncta in the nucleus are CTD-dependent and show a distinct localization pattern.

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Data availability

Statistical source data for Figs. 1–5 and Extended Data Fig. 2–6, 9 and the unprocessed gel image for Extended Data Fig. 1c are provided with this paper. Other data are available upon reasonable request.

Code availability

All specified scripts used to run C-Trap experiments or analyze their results can be accessed on LUMICKS Harbor (https://harbor.lumicks.com/). All custom-written codes will be made available upon request.

References

  1. Woodcock, C. L., Skoultchi, A. I. & Fan, Y. Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length. Chromosome Res 14, 17–25 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Song, F. et al. Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344, 376–380 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Bednar, J. et al. Structure and dynamics of a 197 bp nucleosome in complex with linker histone H1. Mol. Cell 66, 384–397 e8 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Zhou, B. R. et al. Distinct structures and dynamics of chromatosomes with different human linker histone isoforms. Mol. Cell 81, 166–182 e6 (2021).

    Article  CAS  PubMed  Google Scholar 

  5. Rudnizky, S. et al. Extended and dynamic linker histone-DNA Interactions control chromatosome compaction. Mol. Cell 81, 3410–3421 e4 (2021).

    Article  CAS  PubMed  Google Scholar 

  6. Xiao, B., Freedman, B. S., Miller, K. E., Heald, R. & Marko, J. F. Histone H1 compacts DNA under force and during chromatin assembly. Mol. Biol. Cell 23, 4864–4871 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. White, A. E., Hieb, A. R. & Luger, K. A quantitative investigation of linker histone interactions with nucleosomes and chromatin. Sci. Rep. 6, 19122 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Perisic, O., Portillo-Ledesma, S. & Schlick, T. Sensitive effect of linker histone binding mode and subtype on chromatin condensation. Nucleic Acids Res. 47, 4948–4957 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Willcockson, M. A. et al. H1 histones control the epigenetic landscape by local chromatin compaction. Nature 589, 293–298 (2021).

    Article  CAS  PubMed  Google Scholar 

  10. Hergeth, S. P. & Schneider, R. The H1 linker histones: multifunctional proteins beyond the nucleosomal core particle. EMBO Rep. 16, 1439–1453 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fyodorov, D. V., Zhou, B. R., Skoultchi, A. I. & Bai, Y. Emerging roles of linker histones in regulating chromatin structure and function. Nat. Rev. Mol. Cell Biol. 19, 192–206 (2018).

    Article  CAS  PubMed  Google Scholar 

  12. Fan, Y. et al. Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation. Cell 123, 1199–1212 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Torres, C. M. et al. The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity. Science 353, aaf1644 (2016).

  14. Yusufova, N. et al. Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture. Nature 589, 299–305 (2021).

    Article  CAS  PubMed  Google Scholar 

  15. Arimura, Y., Shih, R. M., Froom, R. & Funabiki, H. Structural features of nucleosomes in interphase and metaphase chromosomes. Mol. Cell 81, 4377–4397 (2021).

  16. Catez, F., Ueda, T. & Bustin, M. Determinants of histone H1 mobility and chromatin binding in living cells. Nat. Struct. Mol. Biol. 13, 305–310 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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 

  20. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhang, H. et al. RNA controls PolyQ protein phase transitions. Mol. Cell 60, 220–230 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nott, T. J., Craggs, T. D. & Baldwin, A. J. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters. Nat. Chem. 8, 569–575 (2016).

    Article  CAS  PubMed  Google Scholar 

  23. Vieregg, J. R. et al. Oligonucleotide-peptide complexes: phase control by hybridization. J. Am. Chem. Soc. 140, 1632–1638 (2018).

    Article  CAS  PubMed  Google Scholar 

  24. Shakya, A. & King, J. T. DNA flexibility-dependent assembly of phase-separated liquid droplets. Biophys. J. 115, 1840–1847 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Turner, A. L. et al. Highly disordered histone H1-DNA model complexes and their condensates. Proc. Natl Acad. Sci. USA 115, 11964–11969 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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 

  27. Shakya, A., Park, S., Rana, N. & King, J. T. Liquid-liquid phase separation of histone proteins in cells: role in chromatin organization. Biophys. J. 118, 753–764 (2020).

    Article  CAS  PubMed  Google Scholar 

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

  29. Smith, S. B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).

    Article  CAS  PubMed  Google Scholar 

  30. van Mameren, J. et al. Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc. Natl Acad. Sci. USA 106, 18231–18236 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Alshareedah, I. et al. Interplay between short-range attraction and long-range repulsion controls reentrant liquid condensation of ribonucleoprotein-RNA complexes. J. Am. Chem. Soc. 141, 14593–14602 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rhine, K. et al. ALS/FTLD-linked mutations in FUS glycine residues cause accelerated gelation and reduced interactions with wild-type FUS. Mol. Cell 80, 666–681 e8 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ghosh, A. & Zhou, H. X. Determinants for fusion speed of biomolecular droplets. Angew. Chem. Int. Ed. Engl. 59, 20837–20840 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Savelyev, A. & Papoian, G. A. Chemically accurate coarse graining of double-stranded DNA. Proc. Natl Acad. Sci. USA 107, 20340–20345 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Latham, A. P. & Zhang, B. Consistent force field captures homologue-resolved HP1 phase separation. J. Chem. Theory Comput. 17, 3134–3144 (2021).

  36. Latham, A. P. & Zhang, B. On the stability an d layered organization of protein-DNA condensates. Biophys. J. https://doi.org/10.1016/j.bpj.2022.03.028 (2022).

    Article  PubMed  Google Scholar 

  37. Borgia, A. et al. Extreme disorder in an ultrahigh-affinity protein complex. Nature 555, 61–66 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Espinosa, J. R. et al. Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components. Proc. Natl Acad. Sci. USA 117, 13238–13247 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dignon, G. L., Zheng, W., Best, R. B., Kim, Y. C. & Mittal, J. Relation between single-molecule properties and phase behavior of intrinsically disordered proteins. Proc. Natl Acad. Sci. USA 115, 9929–9934 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Misteli, T., Gunjan, A., Hock, R., Bustin, M. & Brown, D. T. Dynamic binding of histone H1 to chromatin in living cells. Nature 408, 877–881 (2000).

    Article  CAS  PubMed  Google Scholar 

  41. Hendzel, M. J., Lever, M. A., Crawford, E. & Th’ng, J. P. The C-terminal domain is the primary determinant of histone H1 binding to chromatin in vivo. J. Biol. Chem. 279, 20028–20034 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Koc, A., Wheeler, L. J., Mathews, C. K. & Merrill, G. F. Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. J. Biol. Chem. 279, 223–230 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Foote, K. M. et al. Discovery and characterization of AZD6738, a potent inhibitor of ataxia telangiectasia mutated and Rad3 related (ATR) kinase with application as an anticancer agent. J. Med. Chem. 61, 9889–9907 (2018).

    Article  CAS  PubMed  Google Scholar 

  44. Thakar, T. et al. Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly. Nat. Commun. 11, 2147 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang, Y., Xu, B., Weiner, B. G., Meir, Y. & Wingreen, N. S. Decoding the physical principles of two-component biomolecular phase separation. eLife 10, e62403 (2021).

  46. Downs, J. A., Kosmidou, E., Morgan, A. & Jackson, S. P. Suppression of homologous recombination by the Saccharomyces cerevisiae linker histone. Mol. Cell 11, 1685–1692 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Hashimoto, H. et al. Histone H1 variant, H1R is involved in DNA damage response. DNA Repair 6, 1584–1595 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Zheng, Y. et al. Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II. J. Cell Biol. 189, 407–415 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Osunsade, A. et al. A robust method for the purification and characterization of recombinant human histone H1 variants. Biochemistry 58, 171–176 (2019).

    Article  CAS  PubMed  Google Scholar 

  50. Izzo, A. & Schneider, R. The role of linker histone H1 modifications in the regulation of gene expression and chromatin dynamics. Biochim. Biophys. Acta 1859, 486–495 (2016).

    Article  PubMed  CAS  Google Scholar 

  51. Fierz, B. et al. Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction. Nat. Chem. Biol. 7, 113–119 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Li, S., Zheng, E. B., Zhao, L. & Liu, S. Nonreciprocal and conditional cooperativity directs the pioneer activity of pluripotency transcription factors. Cell Rep. 28, 2689–2703 e4 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wasserman, M. R., Schauer, G. D., O’Donnell, M. E. & Liu, S. Replication fork activation is enabled by a single-stranded DNA gate in CMG helicase. Cell 178, 600–611 e16 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Harada, B. T. et al. Stepwise nucleosome translocation by RSC remodeling complexes. eLife 5, e10051 (2016).

  55. Luger, K., Rechsteiner, T. J. & Richmond, T. J. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999).

    Article  CAS  PubMed  Google Scholar 

  56. Lee, K. M. & Narlikar, G. Assembly of nucleosomal templates by salt dialysis. Curr. Protoc. Mol. Biol. https://doi.org/10.1002/0471142727.mb2106s54 (2001).

  57. Schauer, G., Finkelstein, J. & O’Donnell, M. In vitro assays for eukaryotic leading/lagging strand DNA replication. Bio. Protoc. 7, e2548 (2017).

  58. Sbalzarini, I. F. & Koumoutsakos, P. Feature point tracking and trajectory analysis for video imaging in cell biology. J. Struct. Biol. 151, 182–195 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Mangeol, P., Prevo, B. & Peterman, E. J. KymographClear and KymographDirect: two tools for the automated quantitative analysis of molecular and cellular dynamics using kymographs. Mol. Biol. Cell 27, 1948–1957 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Crickard, J. B., Moevus, C. J., Kwon, Y., Sung, P. & Greene, E. C. Rad54 drives ATP hydrolysis-dependent DNA sequence alignment during homologous recombination. Cell 181, 1380–1394 e18 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Harris, C. R. et al. Array programming with NumPy. Nature 585, 357–362 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Savelyev, A. & Papoian, G. A. Chemically accurate coarse graining of double-stranded DNA. Proc. Natl Acad. Sci. USA 107, 20340–20345 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Clementi, C., Nymeyer, H. & Onuchic, J. N. Topological and energetic factors: what determines the structural details of the transition state ensemble and ‘en-route’ intermediates for protein folding? An investigation for small globular proteins. J. Mol. Biol. 298, 937–953 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Sing, C. E. & Perry, S. L. Recent progress in the science of complex coacervation. Soft Matter 16, 2885–2914 (2020).

    Article  CAS  PubMed  Google Scholar 

  65. Berendsen, H. J. C., van der Spoel, D. & van Drunen, R. GROMACS: a message-passing parallel molecular dynamics implementation. Comput. Phys. Commun. 91, 43–56 (1995).

    Article  CAS  Google Scholar 

  66. Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    Article  CAS  PubMed  Google Scholar 

  67. Michaud-Agrawal, N., Denning, E. J., Woolf, T. B. & Beckstein, O. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. J. Comput. Chem. 32, 2319–2327 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Dignon, G. L., Zheng, W., Kim, Y. C., Best, R. B. & Mittal, J. Sequence determinants of protein phase behavior from a coarse-grained model. PLoS Comput. Biol. 14, e1005941 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We thank the O’Donnell laboratory (Rockefeller University) for RPA, A. Soshnev (Rockefeller) for discussions and M. Tipping and Y. Romin (MSK Molecular Cytology core facility) for advice on live-cell imaging and analysis. A.O. and A.P.L. are supported by the National Science Foundation Graduate Research Fellowship. T.N. is supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under award number T32GM007739 to the Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD/PhD Program. B.Z. is supported by the NIH grant no. R35GM133580. Y.D. is supported by NIH grant no. R35GM138386, CCSG core grant no. P30CA008748, the Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research and the Center for Experimental Therapeutics at MSKCC, the Parker Institute for Cancer Immunotherapy and the Anna Fuller Cancer Research Foundation. S.L. is supported by the Robertson Foundation, the Alfred P. Sloan Foundation and an NIH Director’s New Innovator Award (no. DP2HG010510). Y.D. and S.L. also acknowledge support from the Pershing Square Sohn Cancer Research Alliance. Y.D. is a Josie Robertson Young Investigator.

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Author notes

  1. These authors contributed equally: Rachel Leicher, Adewola Osunsade, Gabriella N. L. Chua, Sarah C. Faulkner.

Authors and Affiliations

  1. Laboratory of Nanoscale Biophysics and Biochemistry, The Rockefeller University, New York, NY, USA

    Rachel Leicher, Gabriella N. L. Chua, John W. Watters, Tuan Nguyen & Shixin Liu

  2. Tri-Institutional PhD Program in Chemical Biology, New York, NY, USA

    Rachel Leicher, Adewola Osunsade, Gabriella N. L. Chua, Sophia Christodoulou-Rubalcava, Yael David & Shixin Liu

  3. Chemical Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center (MSKCC), New York, NY, USA

    Adewola Osunsade, Sarah C. Faulkner & Yael David

  4. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Andrew P. Latham & Bin Zhang

  5. Tri-Institutional MD-PhD Program, New York, NY, USA

    Tuan Nguyen, Paul G. Young, Yael David & Shixin Liu

  6. Laboratory of DNA Replication, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA

    Emily C. Beckwitt

  7. Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA

    Yael David

  8. Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medical College, New York, NY, USA

    Yael David

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Contributions

R.L. and G.N.L.C. prepared the DNA samples, performed single-molecule binding, in vitro phase separation and droplet fusion experiments. J.W.W. developed software for in vitro data analysis. T.N. performed the TIRF experiments. E.C.B. constructed the forked DNA substrate. A.O. and S.C.F. prepared the H1 constructs and performed in vivo experiments with help from S.C.-R. and P.G.Y. A.P.L. and B.Z. performed the molecular dynamics simulations. Y.D. and S.L. oversaw the project. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Yael David or Shixin Liu.

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Nature Structural &Molecular Biology thanks Katherine Stott and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Carolina Perdigoto and Beth Moorefield, in collaboration with the Nature Structural &Molecular Biology team.

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Extended data

Extended Data Fig. 1 H1 purification and labeling.

a, Predictor of natural disordered regions (PONDR) score for the H1.4 amino acid sequence (www.pondr.com). A score of >0.5 is considered intrinsically disordered. b, Schematic of the domain structures of full-length H1 and NTD/CTD-truncated H1 constructs. c, Representative SDS-PAGE gel scanned for fluorescence showing purified Cy3-labeled H1.4 (among 5 independent preparations).

Source data

Extended Data Fig. 2 H1 and core histones respond differently to force applied to the DNA tether.

a, A kymograph showing Cy3-H1 binding to λ-DNA over time as the inter-bead distance was increased. b, Total Cy3 intensity across the DNA tether over time for the kymograph shown in a. c, Schematic of the final H1 binding configuration for the example shown in a. d, A representative kymograph showing Cy3-H2B binding to λ-DNA over time as the inter-bead distance was increased. e, Total Cy3 intensity across the DNA tether over time for the kymograph shown in d.

Source data

Extended Data Fig. 3 Reversibility of H1:ssDNA foci formed on tethered DNA.

a, A representative kymograph showing reversible formation and dissolution of Cy3-H1 foci during DNA tether stretching and relaxation. b, A representative kymograph showing the persistence of Cy3-H1 foci after tether relaxation in the presence of AlexaFluor488-RPA. c, Fraction of H1 foci dissolved versus retained in the absence or presence of RPA.

Source data

Extended Data Fig. 4 H1 colocalizes and forms condensates with nucleosomes.

a, Schematic of the total-internal-reflection fluorescence (TIRF) microscopy assay using surface-immobilized λ DNA loaded with Cy5-H2B nucleosomes. b, Schematic of the experimental setup in a after Cy3-H1 is added to the flow chamber. c, Representative fluorescence images (among 3 independent experiments) of Cy5-H2B nucleosomes (top) and Cy3-H1 (bottom) on λ DNA. Scale bar: 0.5 µm. d, Fluorescence intensity profiles of Cy5-H2B and Cy3-H1 over the DNA length for the images in c. e, Snapshots of a representative fusion event for H1:Cy3-H2A mononucleosome droplets visualized by Cy3 fluorescence (among 21 independent fusion events).

Source data

Extended Data Fig. 5 Additional analyses of the biophysical properties of H1:DNA droplets.

a, A representative series of images (among 28 independent experiments) during the photobleaching and fluorescence recovery of an H1:Cy5-ssDNA75 droplet. b, Kinetics of fluorescence recovery for H1:Cy5-ssDNA75 (blue) (n = 28), H1:Cy5-dsDNA75 (red) (n = 15), and H1∆N:ssDNA75 (green) (n = 17) droplets. Data are presented as mean values ± SEM. c, Droplet fusion time (τ) for H1:Cy5-ssDNA75 (n = 56), H1:Cy5-dsDNA75 (n = 24), and H1∆N:ssDNA75 (n = 15) droplets. The top and bottom edges of each box represent the 3rd and 1st quartiles of the data, and the middle line in each box represents the median value. The top and bottom whiskers represent the maximum and minimum values. Significance calculated using a one-way ANOVA with Dunnett’s test for multiple comparisons (***p < 0.001).

Source data

Extended Data Fig. 6 Computational examination of H1:DNA phase separation.

a, Phase behavior of the four simulated systems (H1:ssDNA30, H1:dsDNA30, H1:ssDNA70, H1:dsDNA70) as a function of temperature. b, Probability distribution of the percentage of H1 molecules found in the largest cluster at a temperature of 300 K. c, Probability distribution of the number of DNA molecules bound to each H1 molecule. d, Test of finite size effects on the computational phase diagrams in the temperature-concentration plane. For dsDNA, smaller systems with 40 H1 and 160 DNA molecules were used, but the trends shown in Fig. 3a are conserved. e, Representative configurations for the ssDNA30 system above and below TC. f, A single H1:dsDNA70 pair at different time points in our simulation. g, Different pairs of H1:dsDNA70 at one time point. h, Contact map of bound H1:DNA pairs. Data include all H1:DNA pairs that have at least one residue in contact. The top half of the matrix represents the mean values of the fraction of time each residue-nucleotide pair is in contact, given that the H1 and DNA molecules are in contact. The bottom half represents the standard deviation of this matrix across time and ensemble. The significantly larger standard deviations relative to the mean values support the conformational heterogeneity of H1:dsDNA complexes. i, Diffusion coefficient (D) of H1 in each system at 300 K. All values were normalized by the median D for ssDNA70. Each data point represents the D value for an individual H1 molecule over the course of our simulation (n = 40 for ssDNA, n = 80 for dsDNA). Significance determined using a two-sample t-test (*p < 0.05, ***p < 0.001). The top and bottom edges of each box represent the 3rd and 1st quartiles of the data respectively, and the middle line indicates the median value. Whiskers extend to the data set that are within 2.7σ, where σ is the standard deviation. j, Average number of DNA residues in contact with any given H1 residue as a function of the H1 residue index. k, Average number of DNA residues in contact with any given H1 residue projected onto a structural model of H1. Data range from most contacts (red) to fewest contacts (blue).

Source data

Extended Data Fig. 7 Additional live-cell images of eGFP-H1 and eGFP-H1∆C.

Representative confocal fluorescence images of HEK293T cells transfected with either eGFP-H1 or eGFP-H1∆C and treated with either mock or 2 mM HU + 20 µM AZD6738 for 18 h, and their corresponding brightfield images (among 10 independent nuclei imaged for each condition).

Extended Data Fig. 8 Timepoints from continuous Z-stack monitoring for merging eGFP-H1 puncta.

Representative slices from continuous Z-stack imaging over 10 minutes of HEK293T cells transfected with eGFP-H1 and treated with 2 mM HU + 20 µM AZD6738 for 18 h (among 6 imaging acquisitions on independent nuclei). For each timepoint, top left=x plane, top right=y plane, bottom left=z plane.

Extended Data Fig. 9 Quantification of RPA puncta in the nucleus.

Violin plot showing the distribution of number of RPA puncta per cell for eGFP-H1 and eGFP-H1∆C transfected cells after mock or HU + AZD6738 treatment. Significance calculated using Welch’s t-test (**p < 0.01, ***p < 0.001). eGFP-H1, mock (n = 16 independent nuclei), eGFP-H1ΔC, mock (n = 12), eGFP-H1, treated (n = 15), eGFP-H1ΔC, treated (n = 12).

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Extended Data Fig. 10 H1 interaction with forked DNA in vitro.

a, Schematic of a tethered DNA substrate containing a fork junction near one end of the tether. b, Two representative kymographs showing Cy3-H1 coalescing with relaxed ssDNA towards the fork junction near the bead. A schematic of the H1 binding configuration is shown on the left. Imaging was performed with green laser on. c, A representative kymograph showing AlexaFluor488-RPA binding to ssDNA regions formed by unpeeling from tethered ends and stochastically occurring internal nicks or by melting of dsDNA as the inter-bead distance was increased. Imaging was performed with blue laser on. d, A representative kymograph obtained with Cy3-H1 and AlexaFluor488-RPA showing that H1:ssDNA condensate (white arrow) prevents further force-induced ssDNA unpeeling at the fork junction, which would result in an expansion of RPA-bound ssDNA near the fork region. Imaging was performed with both green and blue lasers on.

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Supplementary Table 1, Figs. 1–9 and Data for Supplementary Figs. 1b–d and 9.

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Leicher, R., Osunsade, A., Chua, G.N.L. et al. Single-stranded nucleic acid binding and coacervation by linker histone H1. Nat Struct Mol Biol 29, 463–471 (2022). https://doi.org/10.1038/s41594-022-00760-4

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