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Nuclear F-actin and myosins drive relocalization of heterochromatic breaks

Nature volume 559, pages 54–60 (2018)Cite this article

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Abstract

Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, ‘safe’ repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.

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Fig. 1: Actin nucleators mediate relocalization of heterochromatic DSBs.
Fig. 2: Nuclear F-actin is required for relocalization.
Fig. 3: Relocalization relies on nuclear myosins.
Fig. 4: Actin nucleators and myosins drive directed motions of heterochromatic DSBs.
Fig. 5: Nuclear F-actin and myosins promote heterochromatin integrity.

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References

  1. Amaral, N., Ryu, T., Li, X. & Chiolo, I. Nuclear dynamics of heterochromatin repair. Trends Genet. 33, 86–100 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Caridi, P. C., Delabaere, L., Zapotoczny, G. & Chiolo, I. And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break. Phil. Trans. R. Soc. Lond. B 372, 20160291 (2017).

    Article  Google Scholar 

  3. Ho, J. W. et al. Comparative analysis of metazoan chromatin organization. Nature 512, 449–452 (2014).

    Article  PubMed  PubMed Central  ADS  CAS  Google Scholar 

  4. Hoskins, R. A. et al. The Release 6 reference sequence of the Drosophila melanogaster genome. Genome Res. 25, 445–458 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Chiolo, I. et al. Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell 144, 732–744 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Chiolo, I., Tang, J., Georgescu, W. & Costes, S. V. Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin. Mutat. Res. 750, 56–66 (2013).

    Article  PubMed  CAS  Google Scholar 

  7. Ryu, T. et al. Heterochromatic breaks move to the nuclear periphery to continue recombinational repair. Nat. Cell Biol. 17, 1401–1411 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Ryu, T., Bonner, M. R. & Chiolo, I. Cervantes and Quijote protect heterochromatin from aberrant recombination and lead the way to the nuclear periphery. Nucleus 7, 485–497 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Li, Q. et al. The three-dimensional genome organization of Drosophila melanogaster through data integration. Genome Biol. 18, 145 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Janssen, A. et al. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. Genes Dev. 30, 1645–1657 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Jakob, B. et al. DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin. Nucleic Acids Res. 39, 6489–6499 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Tsouroula, K. et al. Temporal and spatial uncoupling of DNA double strand break repair pathways within mammalian heterochromatin. Mol. Cell 63, 293–305 (2016).

    Article  PubMed  CAS  Google Scholar 

  13. Andrin, C. et al. A requirement for polymerized actin in DNA double-strand break repair. Nucleus 3, 384–395 (2012).

    Article  PubMed  Google Scholar 

  14. Belin, B. J., Lee, T. & Mullins, R. D. DNA damage induces nuclear actin filament assembly by Formin -2 and Spire-1/2 that promotes efficient DNA repair. eLife 4, e07735 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Sun, M. H. et al. DNA double-strand breaks induce the nuclear actin filaments formation in cumulus enclosed oocytes but not in denuded oocytes. PLoS ONE 12, e0170308 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Mullins, R. D., Heuser, J. A. & Pollard, T. D. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl Acad. Sci. USA 95, 6181–6186 (1998).

    Article  PubMed  ADS  CAS  Google Scholar 

  17. Goley, E. D. & Welch, M. D. The ARP2/3 complex: an actin nucleator comes of age. Nat. Rev. Mol. Cell Biol. 7, 713–726 (2006).

    Article  PubMed  CAS  Google Scholar 

  18. Chuang, C.-H. et al. Long-range directional movement of an interphase chromosome site. Curr. Biol. 16, 825–831 (2006).

    Article  PubMed  CAS  Google Scholar 

  19. Dundr, M. et al. Actin-dependent intranuclear repositioning of an active gene locus in vivo. J. Cell Biol. 179, 1095–1103 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Mehta, I. S., Amira, M., Harvey, A. J. & Bridger, J. M. Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts. Genome Biol. 11, R5 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Roberts, K. L. & Baines, J. D. Actin in herpesvirus infection. Viruses 3, 336–346 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Hatakeyama, H., Nakahata, Y., Yarimizu, H. & Kanzaki, M. Live-cell single-molecule labeling and analysis of myosin motors with quantum dots. Mol. Biol. Cell 28, 173–181 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Amitai, A., Seeber, A., Gasser, S. M. & Holcman, D. Visualization of chromatin decompaction and break site extrusion as predicted by statistical polymer modeling of single-locus trajectories. Cell Reports 18, 1200–1214 (2017).

    Article  PubMed  CAS  Google Scholar 

  24. Caridi, C. P. et al. Quantitative methods to investigate the 4D dynamics of heterochromatic repair sites in Drosophila cells. Methods Enzymol. 601, 359–389 (2018).

    Article  PubMed  Google Scholar 

  25. Li, H., Guo, F., Rubinstein, B. & Li, R. Actin-driven chromosomal motility leads to symmetry breaking in mammalian meiotic oocytes. Nat. Cell Biol. 10, 1301–1308 (2008).

    Article  PubMed  CAS  Google Scholar 

  26. Schrank, B. R. et al. Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature https://doi.org/10.1038/s41586-018-0237-5 (2018).

  27. Goodarzi, A. A. et al. ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin. Mol. Cell 31, 167–177 (2008).

    Article  PubMed  CAS  Google Scholar 

  28. Torres-Rosell, J. et al. The Smc5–Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat. Cell Biol. 9, 923–931 (2007).

    Article  PubMed  CAS  Google Scholar 

  29. van Sluis, M. & McStay, B. A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage. Genes Dev. 29, 1151–1163 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Therizols, P. et al. Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region. J. Cell Biol. 172, 189–199 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Khadaroo, B. et al. The DNA damage response at eroded telomeres and tethering to the nuclear pore complex. Nat. Cell Biol. 11, 980–987 (2009).

    Article  PubMed  CAS  Google Scholar 

  32. Cho, N. W., Dilley, R. L., Lampson, M. A. & Greenberg, R. A. Interchromosomal homology searches drive directional ALT telomere movement and synapsis. Cell 159, 108–121 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Chung, D. K. et al. Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process. Nat. Commun. 6, 7742 (2015).

    Article  PubMed  CAS  Google Scholar 

  34. Churikov, D. et al. SUMO-dependent relocalization of eroded telomeres to nuclear pore complexes controls telomere recombination. Cell Reports 15, 1242–1253 (2016).

    Article  PubMed  CAS  Google Scholar 

  35. Lottersberger, F., Karssemeijer, R. A., Dimitrova, N. & de Lange, T. 53BP1 and the LINC complex promote microtubule-dependent DSB mobility and DNA repair. Cell 163, 880–893 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Nagai, S. et al. Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322, 597–602 (2008).

    Article  PubMed  PubMed Central  ADS  CAS  Google Scholar 

  37. Su, X. A., Dion, V., Gasser, S. M. & Freudenreich, C. H. Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability. Genes Dev. 29, 1006–1017 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Kalocsay, M., Hiller, N. J. & Jentsch, S. Chromosome-wide Rad51 spreading and SUMO-H2A.Z-dependent chromosome fixation in response to a persistent DNA double-strand break. Mol. Cell 33, 335–343 (2009).

    Article  PubMed  CAS  Google Scholar 

  39. Oza, P., Jaspersen, S. L., Miele, A., Dekker, J. & Peterson, C. L. Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery. Genes Dev. 23, 912–927 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. Swartz, R. K., Rodriguez, E. C. & King, M. C. A role for nuclear envelope-bridging complexes in homology-directed repair. Mol. Biol. Cell 25, 2461–2471 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Dion, V., Kalck, V., Horigome, C., Towbin, B. D. & Gasser, S. M. Increased mobility of double-strand breaks requires Mec1, Rad9 and the homologous recombination machinery. Nat. Cell Biol. 14, 502–509 (2012).

    Article  PubMed  CAS  Google Scholar 

  42. Miné-Hattab, J. & Rothstein, R. Increased chromosome mobility facilitates homology search during recombination. Nat. Cell Biol. 14, 510–517 (2012).

    Article  PubMed  CAS  Google Scholar 

  43. Cherbas, L. & Gong, L. Cell lines. Methods 68, 74–81 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Goodarzi, A. A., Noon, A. T. & Jeggo, P. A. The impact of heterochromatin on DSB repair. Biochem. Soc. Trans. 37, 569–576 (2009).

    Article  PubMed  CAS  Google Scholar 

  45. Noon, A. T. et al. 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair. Nat. Cell Biol. 12, 177–184 (2010).

    Article  PubMed  CAS  Google Scholar 

  46. Erhardt, S. et al. Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation. J. Cell Biol. 183, 805–818 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Hudson, A. M. & Cooley, L. A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex. J. Cell Biol. 156, 677–687 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Lee, C. F., Melkani, G. C. & Bernstein, S. I. The UNC-45 myosin chaperone: from worms to flies to vertebrates. Int. Rev. Cell Mol. Biol. 313, 103–144 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Kokai, E. et al. Analysis of nuclear actin by overexpression of wild-type and actin mutant proteins. Histochem. Cell Biol. 141, 123–135 (2014).

    Article  PubMed  CAS  Google Scholar 

  50. Plessner, M., Melak, M., Chinchilla, P., Baarlink, C. & Grosse, R. Nuclear F-actin formation and reorganization upon cell spreading. J. Biol. Chem. 290, 11209–11216 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Krauss, J., López de Quinto, S., Nüsslein-Volhard, C. & Ephrussi, A. Myosin-V regulates oskar mRNA localization in the Drosophila oocyte. Curr. Biol. 19, 1058–1063 (2009).

    Article  PubMed  CAS  Google Scholar 

  52. Cheeseman, I. M. & Desai, A. A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 2005, pl1 (2005).

    PubMed  Google Scholar 

  53. Verboon, J. M., Sugumar, B. & Parkhurst, S. M. Wiskott-Aldrich syndrome proteins in the nucleus: aWASH with possibilities. Nucleus 6, 349–359 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Verboon, J. M. et al. Wash interacts with lamin and affects global nuclear organization. Curr. Biol. 25, 804–810 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Larracuente, A. M. & Ferree, P. M. Simple method for fluorescence DNA in situ hybridization to squashed chromosomes. J. Vis. Exp. 95, 52288 (2015).

    Google Scholar 

  56. Small, J. V., Herzog, M. & Anderson, K. Actin filament organization in the fish keratocyte lamellipodium. J. Cell Biol. 129, 1275–1286 (1995).

    Article  PubMed  CAS  Google Scholar 

  57. Sawasdichai, A., Chen, H. T., Abdul Hamid, N., Jayaraman, P. S. & Gaston, K. In situ subcellular fractionation of adherent and non-adherent mammalian cells. J. Vis. Exp. 41, 1958 (2010).

    Google Scholar 

  58. Whelan, D. R. & Bell, T. D. Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters. Sci. Rep. 5, 7924 (2015).

    Article  PubMed  PubMed Central  ADS  CAS  Google Scholar 

  59. Pierobon, P. et al. Velocity, processivity, and individual steps of single myosin V molecules in live cells. Biophys. J. 96, 4268–4275 (2009).

    Article  PubMed  PubMed Central  ADS  CAS  Google Scholar 

  60. Leyton Puig, D. et al. Tips and tricks for artifact-free PFA-based fixation of the actin cytoskeleton and its regulatory proteins for single molecule localization super-resolution microscopy. Protoc. Exch. (2016).

  61. Baarlink, C., Wang, H. & Grosse, R. Nuclear actin network assembly by formins regulates the SRF coactivator MAL. Science 340, 864–867 (2013).

    Article  PubMed  ADS  CAS  Google Scholar 

  62. Yarrow, J. C., Lechler, T., Li, R. & Mitchison, T. J. Rapid de-localization of actin leading edge components with BDM treatment. BMC Cell Biol. 4, 5 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Islam, K. et al. A myosin V inhibitor based on privileged chemical scaffolds. Angew. Chem. 49, 8484–8488 (2010).

    Article  CAS  Google Scholar 

  64. Nolen, B. J. et al. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature 460, 1031–1034 (2009).

    Article  PubMed  PubMed Central  ADS  CAS  Google Scholar 

  65. Belin, B. J., Cimini, B. A., Blackburn, E. H. & Mullins, R. D. Visualization of actin filaments and monomers in somatic cell nuclei. Mol. Biol. Cell 24, 982–994 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Strober, W. Trypan blue exclusion test of cell viability. Curr. Protoc. Immunol. 111, A3.B.1–A3.B.3 (2015).

    Article  Google Scholar 

  67. Liu, R. et al. Wash functions downstream of Rho and links linear and branched actin nucleation factors. Development 136, 2849–2860 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  68. Rodriguez-Mesa, E., Abreu-Blanco, M. T., Rosales-Nieves, A. E. & Parkhurst, S. M. Developmental expression of Drosophila Wiskott–Aldrich Syndrome family proteins. Dev Dyn. 241, 608–626 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  69. Van Hatten, R. A. et al. The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication. J. Cell Biol. 159, 541–547 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. James, T. C. & Elgin, S. C. Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene. Mol. Cell. Biol. 6, 3862–3872 (1986).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  71. Ren, C., Webster, P., Finkel, S. E. & Tower, J. Increased internal and external bacterial load during Drosophila aging without life-span trade-off. Cell Metab. 6, 144–152 (2007).

    Article  PubMed  CAS  Google Scholar 

  72. Quinlan, M. E., Heuser, J. E., Kerkhoff, E. & Mullins, R. D. Drosophila Spire is an actin nucleation factor. Nature 433, 382–388 (2005).

    Article  PubMed  ADS  CAS  Google Scholar 

  73. Afshar, K., Stuart, B. & Wasserman, S. A. Functional analysis of the Drosophila diaphanous FH protein in early embryonic development. Development 127, 1887–1897 (2000).

    PubMed  CAS  Google Scholar 

  74. Melak, M., Plessner, M. & Grosse, R. Actin visualization at a glance. J. Cell Sci. 130, 525–530 (2017).

    Article  PubMed  Google Scholar 

  75. Schoenenberger, C. A. et al. Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm. J. Struct. Biol. 152, 157–168 (2005).

    Article  PubMed  CAS  Google Scholar 

  76. Dopie, J., Skarp, K. P., Rajakylä, E. K., Tanhuanpää, K. & Vartiainen, M. K. Active maintenance of nuclear actin by importin 9 supports transcription. Proc. Natl Acad. Sci. USA 109, E544–E552 (2012).

    Article  PubMed  Google Scholar 

  77. Senaratne, T. N., Joyce, E. F., Nguyen, S. C. & Wu, C. T. Investigating the interplay between sister chromatid cohesion and homolog pairing in Drosophila nuclei. PLoS Genet. 12, e1006169 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

Work supported by USC Gold Family and Research Enhancement Fellowships to T.R., and R01GM117376, Mallinckrodt Foundation Award, and NSF Career 1751197 to I.C. We thank S. Keagy, O. Aparicio and the Chiolo Laboratory for comments; O. Aparicio, M. Michael and V. Longo for reagents and cells; B. Mellone, G. Posern and S. Celniker for plasmids; L. Cooley, A. Eprussi, H. Ohkura, M. Gatti, S. Bernstein, G. Karpen, J. Kadonaga, S. Elgin, S. Parkhurst and DSHB for antibodies; F. Cuturale, C. Artesano, S. Fraser and the Translation Imaging Center for help with imaging and analyses; H. Hopp, B. Spatola, P. Palagani and A. Holcom for experimental help; and the BDSC (P40OD018537), VDRC, and DGRC (P40OD010949) for flies and plasmids. We apologize to our colleagues whose work could not be cited owing to space limitations.

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  1. Nuno Amaral

    Present address: Karolinska Institute, Stockholm, Sweden

  2. These authors contributed equally: Christopher P. Caridi, Carla D’Agostino.

Authors and Affiliations

  1. Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA, USA

    Christopher P. Caridi, Carla D’Agostino, Taehyun Ryu, Grzegorz Zapotoczny, Laetitia Delabaere, Xiao Li, Varandt Y. Khodaverdian, Nuno Amaral, Emily Lin, Alesandra R. Rau & Irene Chiolo

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Contributions

C.P.C. and C.D. performed most experiments; T.R. performed experiments for Figs. 1e, 3a, c, 5c–e and Extended Data Figs. 1f, g, 2i, 4i, 5d, 6c, h, 8c; G.Z. for Figs. 1b, 3a, 5b, e and Extended Data Figs. 1k, 2f, 6i; L.D. for Extended Data Figs. 3e, 8h and complementary studies; X.L. for Fig. 5f and Extended Data Fig 8h; V.Y.K for Fig. 3a and Extended Data Figs. 1g, 6b, h, 8a–c, f; N.A. for Fig. 2a, d; E.L. for Fig. 5c; and A.R.R. for Extended Data Fig. 8h. I.C. conceived and supervised the project, contributed to experiments and wrote the manuscript.

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Correspondence to Irene Chiolo.

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Extended data figures and tables

Extended Data Fig. 1 Actin nucleators mediate relocalization of heterochromatic DSBs.

a, Immunofluorescence and quantification of γH2Av foci in DAPI-bright heterochromatin (dashed circle), or total focus number, of Kc cells fixed 60 min after IR after treatment with LatB (+) or control (Ctrl, −). ****P < 0.0001, ***P = 0.0008 versus control, n > 300 cells per treatment. b, Western blotting or qPCR analyses show RNAi depletion efficiencies for indicated proteins in Kc cells. Actin or a background band (*) were used as loading controls. c, Quantification of total γH2Av foci from Fig. 1a. d, Immunofluorescence and quantification of γH2Av foci at the nuclear periphery (Lamin), or total focus number, 60 min after IR after indicated RNAi depletions in Kc cells. Only middle z stacks were used for quantifications, as previously described7. ****P < 0.0001, n > 200 cells per RNAi. e, As in a but for cells treated with CK666. ****P < 0.0001, *P = 0.0293, n > 300 cells per treatment. f, g, Quantification of γH2Av foci in DAPI-bright heterochromatin or total focus number, after 60 min treatment with, and release from, LatB or CK666. ****P < 0.0001, n > 200 cells per condition. h, Quantification of γH2Av foci in DAPI-bright heterochromatin, or total focus number, in Kc cells fixed 60 min after IR, after RNAi depletion of actin nucleators Spire72 or the formin Dia73. ****P < 0.0001 versus control, n > 300 cells per RNAi. i, qPCR analysis shows ARP3 RNAi depletion efficiency in NIH3T3 cells. j, Quantification of total γH2AX foci for Fig. 1b. k, Quantification of γH2AX foci in DAPI-bright heterochromatin, or total focus number, in NIH3T3 G2 cells treated with indicated chemicals, and fixed 60 min after IR. ****P < 0.0001 versus control, n > 37 cells per treatment. l, Quantification of total γH2Av foci for Fig. 1c. Scale bars, 1 µm. Data shown as mean ± s.e.m. in c, j and mean ± s.d. of at least three independent experiments in a, d–h, k, l. P values calculated using two-tailed Mann–Whitney test.

Source Data

Extended Data Fig. 2 Actin nucleators mediate relocalization of heterochromatic DSBs.

a, Images 10 min after IR and quantification before (−) and 10 min after (+) IR of Mdc1 and Atrip foci in cells expressing GFP–Mdc1 or GFP–Atrip and mCherry–HP1a, after indicated RNAi depletions. n ≥ 48 cells for Atrip; n ≥ 21 cells for Mdc1. b, Quantification of Rad51 foci in cells fixed 60 min after IR and processed for immunofluorescence with anti-Rad51 antibodies after indicated RNAi depletions. Average number of foci shown relative to DAPI-bright or total foci. Smc5/6 RNAi results in abnormal formation of Rad51 foci in heterochromatin5,7,8, and is used as positive control. ****P < 0.0001 versus control, unpaired t-test with Welch’s correction, n > 300 cells per RNAi. c, Images and quantifications of Smc6 and Nse2 colocalizing with the HP1a domain before (−) IR and forming foci before and 15 min after (+) IR in cells expressing GFP–Smc6 or GFP–Nse2 and mCherry–HP1a after indicated RNAi depletions. n = 35 cells for Nse2 and n ≥ 23 cells for Smc6. d, Quantification 30 min after IR of γH2Av foci colocalizing with FHA-tagged Arp2 or with Arpc3B after indicated RNAi depletions. ****P < 0.0001, two-tailed Mann–Whitney test, n ≥ 31 cells per RNAi. e, Quantification of total foci for Fig. 1d. f, Western blot analyses show RNAi depletion efficiency for Arpc3B, and specificity of anti-Arp3cB antibodies. Tubulin is used as loading control. g, Immunofluorescence of γH2Av foci colocalizing with Arpc3B and quantification of total focus number for Fig. 1f. h, Quantification at indicated times after IR of the percentage of γH2Av foci colocalizing with FHA–Arp2 relative to H3K9me3 n = 30. i, qPCR analysis shows Mre11 RNAi efficiency. Scale bars, 1 µm. Data shown as mean ± s.e.m. in a, c, d, g, h and mean ± s.d. of at least three independent experiments in b, e.

Source Data

Extended Data Fig. 3 Nuclear F-actin is required for relocalization.

a, Western blot showing nuclear and cytoplasmic actin levels in cells stably transfected with F-actCB–GFP–NLS. HP1a and tubulin were used as loading controls and to check for purity of the fractions. Consistent with previous studies, expression of F-actCB–GFP–NLS does not alter nuclear actin levels50,74, providing a non-invasive method for nuclear actin detection in vivo. b, Frames of time-lapse experiments (Fig. 2a, Supplementary Videos 1, 2) of cells expressing F-actCB–GFP–NLS and mCherry–HP1a signals treated (+) or not treated (−) with IR. Time points are from IR (+IR Video) or the start of imaging (−IR Video). c, Immunofluorescence and quantification of cells stained with phalloidin show the formation of canonical nuclear actin filaments75 (highlighted in red) at indicated time points after IR. ****P < 0.0001 for 10 min versus 0 min or 30 min, two-tailed Mann–Whitney test, n ≥ 51 cells per time point. Error bars show s.d. of at least three independent experiments. d, Immunofluorescence and staining for GFP, phalloidin, and γH2Av, of cells expressing F-actCB–GFP–NLS, show examples of actin filaments coated with damage foci in the absence (−) of triton extraction (TE), and actin puncta colocalizing with γH2Av foci after (+) TE. Zoomed details highlight colocalizations. e, Immunofluorescence and staining for HA and phalloidin (Phall) of cells expressing FHA–Arp2 show enrichment of Arp2 along nuclear actin filaments, including at the base of actin branches (arrowheads). f, Measurement of F-actin branch angles in response to IR, in time-lapse experiments with cells expressing F-actCB–GFP–NLS. n = 54 angles. g, Quantification of the average duration of IR-induced actin filaments in time-lapse experiments with cells expressing F-actCB–GFP–NLS. n = 162 actin filaments. Dashed red lines indicate median values in f, g. Scale bars, 1 µm.

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Extended Data Fig. 4 Nuclear F-actin is required for relocalization.

a, Frames from time-lapse experiment show cells expressing F-actCB–GFP–NLS and Mdc1–mCherry (Fig. 2c, Supplementary Video 3) with an example of a Mdc1 focus moving along an actin filament. Time indicates minutes from beginning of focus movement along the filament. Mdc1 focus tracking (bottom panels) was done in Imaris. b, Selected frames from cells expressing F-actCB–GFP–NLS and Mdc1–mCherry or HP1a–mCherry show examples of filament directionality at indicated time points after IR, that is, filaments emerging from the heterochromatin domain periphery (top), from repair foci (middle) or from the nuclear periphery (bottom). Percentages indicate the frequency with which each behaviour is observed. n ≥ 44 filaments. c, Quantification of the length of actin filaments departing from the heterochromatin domain relative to the average distance between the HP1a domain periphery and the nuclear periphery. n > 140 filaments. Dashed red vertical line shows median value. d, Immunofluorescence and staining for Flag (actin(WT)), phalloidin (Phall), and γH2Av, of cells expressing Flag–NLS–actin(WT), show examples of actin signals in cells processed by triton extraction before fixation, 10 min after IR. Zoomed details highlight colocalizations. Scale bar, 1 µm. e, Immunofluorescence of cells expressing Flag–NLS-tagged versions of actin(R62D) and actin(S14C) shows colocalizations with γH2Av foci. Quantification shows total γH2Av foci for Fig. 2d. f, Quantification of γH2Av foci colocalizing with Flag–NLS–actin (WT) 10 min after IR, or total number of foci, after indicated RNAi depletions. ****P < 0.0001, n ≥ 14 cells and n ≥ 158 foci per RNAi. g, qPCR analysis shows Ipo9 RNAi depletion efficiency. Ipo9 depletion specifically blocks the transfer of actin monomers to the nucleus14,76. h, Quantification of total γH2Av foci for Fig. 2e. i, Quantification of cells fixed 60 min after IR shows γH2Av foci at the nuclear periphery (lamin), or total focus number, after expression of indicated actin forms. Only the middle z stack was used for quantifications. ****P < 0.0001, n ≥ 215 cells per experiment. Error bars, s.e.m. in e, f, h and s.d. of three independent experiments in i. P values calculated with two-tailed Mann–Whitney test.

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Extended Data Fig. 5 Relocalization relies on nuclear myosins.

a, Western blot analysis of cells expressing FHA-tagged components as indicated, shows RNAi depletion efficiency for indicated proteins. Background bands used as loading controls are indicated by an asterisk. b, Quantification shows total γH2Av foci for Fig. 3a. c, Quantification of γH2Av foci at the nuclear periphery (lamin) or total focus number in cells fixed 60 min after IR, after indicated RNAi depletions. ****P < 0.0001 versus control, n ≥ 200 cells per RNAi. Only the middle Z-stack was used for quantifications. Control RNAi as in Extended Data Fig. 1d. d, Quantification of γH2Av foci in DAPI-bright heterochromatin, or total focus number, in cells fixed 60 min after IR, after indicated RNAi depletions. ****P < 0.0001, ***P = 0.003, **P = 0.0022, *P ≤ 0.0402 versus control, n ≥ 200 cells per RNAi. e, Quantification of total γH2Av foci for Fig. 3b. f, Images 10 min after IR and quantification at 0 (−IR) and 10 min after (+) IR of Mdc1 and Atrip foci in cells expressing GFP–Mdc1 or GFP–Atrip and mCherry–HP1a, after indicated RNAi depletions. n ≥ 75 cells for Atrip and n ≥ 17 cells for Mdc1. g, Images and quantifications of Smc6 and Nse2 colocalizing with the mCherry–HP1a domain before (−) IR and forming foci at 0 and 15 min after (+) IR in cells expressing GFP–Smc6 or GFP–Nse2 and mCherry–HP1a after indicated RNAi depletions. n ≥ 31 cells for Nse2 and n ≥ 21 cells for Smc6. In f, g, Myo indicates RNAi depletion of Myo1A, Myo1B and MyoV. Scale bars, 1 µm. Data shown as mean ± s.e.m. in b, f, g and mean ± s.d. of at least three independent experiments in c–e. All P values calculated with two-tailed Mann–Whitney test except one-tailed Mann–Whitney test was used for Myo1B RNAi in d.

Source Data

Extended Data Fig. 6 Relocalization relies on nuclear myosins.

a, Quantification of Rad51 foci in cells fixed 60 min after IR after indicated RNAi depletions shows the average number of foci relative to DAPI-bright heterochromatin or total foci. ****P = 0.0001 versus control, n > 300 cells per RNAi. Control and Smc5/6 RNAi as in Extended Data Fig. 2b. b, Western blot validation of the antibodies used in c and in Fig. 3c shows loss of Rad50, TopBP1 and dPIAS bands after corresponding RNAi depletions. c, Western blot analysis of the same immunoprecipitation shown in Fig. 3c probed with antibodies for dPIAS or TopBP1, with actin as the loading control. d, Quantification 30 min after IR of γH2Av foci colocalizing with FHA–Myo1A, GFP–Myo1B, MyoV or Unc45 after indicated RNAi depletions. ****P < 0.0001, n ≥ 21 cells per RNAi. e, Immunofluorescence 10 min after IR and quantification at indicated time points after IR of γH2Av foci colocalizing with FHA–Myo1A, GFP–Myo1B, MyoV or Unc45. f, Quantification at indicated times after IR of the percentage of foci of γH2Av + FHA–Myo1A, GFP–Myo1B, MyoV or Unc45, colocalizing with H3K9me3. n = 30 cells per experiment per time point. g, Quantification of Kc cells fixed 60 min after IR shows γH2Av foci in DAPI-bright heterochromatin, or total focus number, after treatment with BDM, MyoVin or in controls. ****P < 0.0001, **P < 0.008, n ≥ 300 cells per treatment. h, Quantification of γH2Av foci in DAPI-bright heterochromatin or total foci, after 60 min treatment with and release from BDM or MyoVin. ****P < 0.0001, n ≥ 190 Kc cells per treatment. i, As in g, except NIH3T3 cells were used and γH2AX foci were quantified. Control RNAi as in Extended Data Fig. 1k. ****P < 0.0001, n ≥ 37 cells and n ≥ 2,760 foci per treatment. Data shown as mean ± s.e.m. in d–f and mean ± s.d. of at least three independent experiments in a, g–i. All P values calculated with two-tailed Mann–Whitney test.

Source Data

Extended Data Fig. 7 Actin nucleators and myosin drive directed motions of heterochromatic DSBs.

a, Selected images and quantification at indicated time points of Mdc1 foci in cells expressing GFP–Mdc1 and mCherry–HP1a show no effect of the imaging conditions used in Fig. 4 on the ability of cells to divide or the total number of repair foci. Only representative time points are shown. n = 82 cells. Scale bar, 1 µm. b, Left and middle, quantification and violin plot display of the frequency of heterochromatic Mdc1 foci moving relative to the HP1a domain during the time-lapse experiments shown in Fig. 4, after indicated RNAi depletions. White box, median; red line, mean; vertical black lines, values that occur 95% and 50% of the time. ‘Dynamic HC foci’ include foci moving from inside the HP1a domain to: the nuclear periphery (imNP); outside the HP1a domain (imo); or the periphery of the HP1a domain (imp). ‘Static HC foci’ include foci remaining inside (isi) or at the periphery of the HP1a domain (psp) throughout the 1-h timecourse. Colours reflect the categories analysed in Fig. 4b. Right, quantification of the average number of Mdc1 foci from Fig. 4a–d and b, after indicated RNAi depletions. *P < 0.05, **P < 0.006, ***P = 0.0002; ****P < 0.0001, two-tailed Mann–Whitney test, n ≥ 217 foci per RNAi. c, Quantification of the percentage of heterochromatic (HC) and euchromatic (EU) Mdc1 foci moving to the nuclear periphery in time-lapse experiments from Fig. 4a. ****P < 0.0001; two-tailed Mann–Whitney test, n ≥ 57 foci. d, LDM analysis of Mdc1 foci that reach the nuclear periphery from Fig. 4b. Each coloured horizontal bar represents a focus and its duration in each nuclear compartment, as indicated. The black segments under each bar are LDMs for each focus. n = 28 foci. e, MSD curves generated from the positional data corresponding to the time points that contain LDMs in d. Longer (left) and shorter (right) LDMs are presented as independent graphs for clarity. f, Quantification of the analysis shown in Fig. 4c and d shows the average duration of directed motions (LDMs) or non-directed motions in the nuclear locations defined in d. P < 0.0001 for directed motions in 3 versus 1, 2, 4, unpaired t-test. g, Quantification of LDM durations for euchromatic foci shown in Fig. 4d, after indicated RNAi depletions. h, Quantification and violin plot of Mdc1 focus clustering events in time-lapse experiments for Fig. 4d. Graphical display as in b. An example of clustering is highlighted in Supplementary Video 4. ***P = 0.0003, two-tailed Mann–Whitney test, n ≥ 20 cells per RNAi. Error bars, s.e.m in a, b (right graph), c, f, g.

Source Data

Extended Data Fig. 8 Actin nucleators and myosins promote heterochromatin repair and stability.

a, Western blotting and qPCR analysis show RNAi depletion efficiency for Rad21 and Slmb as indicated. Tubulin is a loading control. b, FISH analysis and quantification show the effect of indicated RNAi on the number of cells with ≥ 3 AACAC or 359bp satellites, reflecting disruption of homologous and/or sister pairing77. **P = 0.0472; ****P < 0.0001, two-tailed Mann–Whitney test, n > 120 cells per RNAi. c, Quantification shows γH2Av foci associated (+) or not associated (−) with H3K9me2 signals 20 h after IR following indicated RNAi depletions. **P = 0.0015; ****P < 0.0001, two-tailed Mann–Whitney test, n > 420 cells per RNAi. d, Quantification of Kc cells from the experiment in Fig. 5a shows γH2Av foci not associated (−) with H3K9me2 signals 20 h after IR and following indicated RNAi depletions. e, Quantification of NIH3T3 cells from the experiment in Fig. 5b shows γH2AX foci not associated (−) with DAPI-bright signals 16 and 24 h after IR and following indicated RNAi depletions. f, Quantification of micronuclei in non-irradiated Kc cells from Fig. 5d. g, Quantification of micronuclei in non-irradiated NIH3T3 cells from Fig. 5e. h, qPCR and western blotting analyses show RNAi depletion efficiency of indicated components in third instar larvae for i and Fig. 5f. i, Images and quantification of chromosomal aberrations in karyotypes from Fig. 5f. Images show extra satellites (arrows), chromosome fusions (arrowhead) and chromosome arm losses (dashed circles). The diagram of Drosophila chromosomes indicates the positions of the main satellites detected by FISH. Aneuploidies and chromosome fusions from Fig. 5f were further categorized to highlight rearrangements involving centromeric regions or chromosomes that are predominantly heterochromatic (fourth or Y). ****P ≤ 0.0001, **P ≤ 0.0079, *P ≤ 0.0433, unpaired t-test with Welsh correction. Error bars, s.e.m. in b, d (RNAi), e, i and s.d. of at least three independent experiments in c, d (actin), f, g.

Source Data

Supplementary information

Supplementary Figure 1

Uncropped scans of the western blots for all figures, as indicated. (*) indicates background bands used as loading controls. (**) indicates signal remaining from a previous hybridization.

Reporting Summary

Supplementary Table 1

Defective relocalization of heterochromatic breaks results in chromosome abnormalities. Quantification of chromosome abnormalities in neuroblasts of 3rd instar larvae for Fig. 5f and Extended Data Fig. 8i.

Supplementary Table 2

RNAi depletion of Rad21 and Slmb affects sister chromatid cohesion and homologous pairing at satellite sequences. Quantification of satellite signals by FISH in Kc cells after indicated RNAi depletions for Extended Data Fig. 8b.

Video 1: F-actin chromobody expression in the nucleus detects a diffuse signal in non-irradiated cells.

Live cell imaging of Kc cells expressing F-actCB-GFP-NLS (F-Actin) and mCh-HP1a. Left panels show F-Actin and HP1a. Right panels show only the F-Actin channel in grey. hh:mm indicates time after the start of imaging.

Video 2: F-actin chromobody expression in the nucleus detects actin filaments in response to IR.

Live cell imaging of Kc cells treated with 5 Gy IR and expressing F-actCB-GFP-NLS (F-Actin) and mCh-HP1a. Left panels show F-Actin and HP1a. Right panels show only the F-Actin channel to highlight actin filaments. hh:mm indicates time after the start of IR treatment.

Video 3: Repair foci move along nuclear actin filaments.

Live cell imaging of Kc cells treated with IR and expressing F-actCB-GFP-NLS (F-Actin) and mCh-Mu2/Mdc1. Left panels show F-Actin and Mu2/Mdc1. Right panels show only the F-Actin channel to highlight actin filaments and the tracking done with Imaris of a repair focus that translocates along a nuclear actin filament. mm:ss indicates time after the start of the movement of the highlighted focus.

Video 4: Heterochromatic repair foci relocalize to the nuclear periphery, while euchromatic repair foci display limited mobility.

Live cell imaging of a Kc cell treated with IR and expressing mCh-HP1a and GFP-Mdc1/Mu2. Cells have been registered with Imaris before focus tracking. Left panels show the HP1a domain (magenta) and Mdc1/Mu2 foci (grey). Four tracked foci are also shown as an example: two originating from the heterochromatin domain (HC, yellow), and two originating from the euchromatic space (EU, cyan), with tracks and volume reconstructions done with Imaris overlaid on the original focus signal. Right panels show only the HP1a domain and the full track of the four foci. This cell corresponds to the example shown in Fig. 5e, where only 3D reconstructions and tracks done with Imaris were shown. hh:mm:ss indicates time after IR. Tracks highlight the more extensive space explored by heterochromatic foci that reach the nuclear periphery relative to euchromatic foci. Tracks also show an example of an euchromatic focus cluster that splits into two foci at 00:51:20 time point.

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Caridi, C.P., D’Agostino, C., Ryu, T. et al. Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature 559, 54–60 (2018). https://doi.org/10.1038/s41586-018-0242-8

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