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

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.

Data availability

Statistical source data for Figs. 15 and Extended Data Fig. 26, 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.

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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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Authors and Affiliations

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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.

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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).

Source data

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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