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Histone degradation in response to DNA damage enhances chromatin dynamics and recombination rates

Nature Structural & Molecular Biology volume 24, pages 99107 (2017) | Download Citation

Abstract

Nucleosomes are essential for proper chromatin organization and the maintenance of genome integrity. Histones are post-translationally modified and often evicted at sites of DNA breaks, facilitating the recruitment of repair factors. Whether such chromatin changes are localized or genome-wide is debated. Here we show that cellular levels of histones drop 20–40% in response to DNA damage. This histone loss occurs from chromatin, is proteasome-mediated and requires both the DNA damage checkpoint and the INO80 nucleosome remodeler. We confirmed reductions in histone levels by stable isotope labeling of amino acids in cell culture (SILAC)-based mass spectrometry, genome-wide nucleosome mapping and fluorescence microscopy. Chromatin decompaction and increased fiber flexibility accompanied histone degradation, both in response to DNA damage and after artificial reduction of histone levels. As a result, recombination rates and DNA-repair focus turnover were enhanced. Thus, we propose that a generalized reduction in nucleosome occupancy is an integral part of the DNA damage response in yeast that provides mechanisms for enhanced chromatin mobility and homology search.

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Acknowledgements

M.H.H. thanks S. Koren-Hauer for critical reading and assistance in preparing the manuscript, and V. Dion and H. Ferreira for fruitful discussions and advice. We thank V. Dion (Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland; strain GA-5816), J. Haber (Department of Biology and Rosenstiel Medical Center, Brandeis University, Waltham, Massachusetts, USA; strain JKM-179), B. Luke (Institute of Molecular Biology, Mainz, Germany; strain GA-3364), K. Bystricky (University of Toulouse, Toulouse, France; precursor strain for GA-9777, strain GA-9227), J.-M. Galan (Institut Jacques Monod, Paris, France; strains GA-1364, GA-1365 and GA-1366) and F. Winston (Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA; plasmids 3494 and 3495) for reagents and material. We are grateful for the technical assistance provided by L. Gelman (microscopy), S. Bourke (microscopy) and H. Kohler (FACS). M.H.H. thanks the Bioinformatics facility for help in getting started with R. We thank all members of the FMI Protein Analysis and Microscopy facilities for valuable advice and support. We thank all members of the Gasser laboratory for valuable discussions and technical support. M.H.H. was supported by a PhD fellowship of the Boehringer Ingelheim Fonds. S.M.G. thanks the HFSP, SNSF and the Novartis Research Foundation for support.

Author information

Author notes

    • Assaf Amitai

    Present address: Institute for Medical Engineering & Science, The Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Michael H Hauer
    • , Andrew Seeber
    • , Raphael Thierry
    • , Ragna Sack
    • , Mariya Kryzhanovska
    • , Jan Eglinger
    •  & Susan M Gasser
  2. Faculty of Natural Sciences, University of Basel, Basel, Switzerland.

    • Michael H Hauer
    • , Andrew Seeber
    •  & Susan M Gasser
  3. Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK.

    • Vijender Singh
    •  & Tom Owen-Hughes
  4. Institut de Biologie de l'École Normale Supérieure, Ecole Normale Supérieure, Paris, France.

    • Assaf Amitai
    •  & David Holcman

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Contributions

M.H.H. and S.M.G. wrote the manuscript. M.H.H. designed experiments and analyzed the data. M.H.H. performed most of the experiments. A.S. contributed to experimental design, data analysis and writing of the manuscript, and performed high-speed live-cell tracking after Zeocin treatment. M.H.H. planned and M.H.H. and A.S. performed the ectopic integration assays and the Rad52-YFP recovery assay. M.K. assisted in ectopic recombination assays. V.S. and T.O.-H. performed and analyzed genome-wide nucleosome mapping. A.A. and D.H. performed biophysical analysis of high-speed tracking data. R.T. performed and maintained the coding for 3D SIM-data analysis. R.S. performed all mass spectrometry measurements and the analysis of label-free experiments. J.E. performed and maintained the coding of tools for 3D interspot distance measurements. All the authors discussed the data and participated in the preparation of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Susan M Gasser.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7, Supplementary Tables 1–4 and Supplementary Note 1

  2. 2.

    Supplementary Dataset 1: Uncropped immunoblot images

    Uncropped blot images used in Figs. 1b, 2a, 2b, 2c and 7a.

Excel files

  1. 1.

    Supplementary Dataset 2: Summary of mobility parameters

    Table showing the strains, conditions and mobility parameters.

  2. 2.

    Supplementary Dataset 4: MS search results: peptides, cycling cells, label swap

    File contains MaxQuant search results used for quantifications.

  3. 3.

    Supplementary Dataset 5: MS search results: protein groups, cycling cells, label swap

    File contains MaxQuant search results used for quantifications.

  4. 4.

    Supplementary Dataset 6: MS search results: peptides, cycling cells, non–label swap

    File contains MaxQuant search results used for quantifications.

  5. 5.

    Supplementary Dataset 7: MS search results: protein groups, cycling cells, non–label swap

    File contains MaxQuant search results used for quantifications.

  6. 6.

    Supplementary Dataset 8: MS search results: peptides, G1 arrest cells

    File contains MaxQuant search results used for quantifications.

  7. 7.

    Supplementary Dataset 9: MS search results: protein groups, G1 arrest cells

    File contains MaxQuant search results used for quantifications.

Zip files

  1. 1.

    Supplementary Dataset 3: KNIME workflow

    File contains the KNIME workflow used for imaging data analysis.

Videos

  1. 1.

    H2B-CFP intensity decreases in response to DNA damage.

    Visualization of data shown in Fig. 1d. Exemplary time course of 9 individual cells following H2B-CFP intensities after 60 min (20–80-min time point) treatment with 300 μg/ml Zeocin for a total time of 120 min. Shown is a merge of Brightfield (average intensity projections) and CFP (maximum intensity projection) channels. Time-lapse series (120 min total) of 100 optical slices per stack (200-nm intervals) were acquired for 12 time points at 10-min intervals, with each slice being exposed for 10 ms per laser line. Video was generated with Fiji (ImagJ) and is shown at 2 frames per second. Original Δt is shown in the top right corner.

  2. 2.

    CFP-LacI and TetR-mRFP time course used for live-cell 3D interdistance measurements.

    Visualization of data shown in Fig. 4b. Exemplary time course of CFP-LacI and TetR-mRFP used for 3D interdistance measurements in living cells. The fluorescent channels were acquired simultaneously on two different CCD cameras, taking 8 optical slices (200-nm thickness) per stack every 300 ms for 2 min, with 10-ms exposure times per slice. Video was generated using the Imaris 8.2.0 software and is shown at 25 frames per second (7.5× faster than the original acquisition speed).

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DOI

https://doi.org/10.1038/nsmb.3347

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