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Nuclear F-actin and myosins drive relocalization of heterochromatic breaks

Naturevolume 559pages5460 (2018) | Download Citation

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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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.

Author information

Author notes

    • Nuno Amaral

    Present address: Karolinska Institute, Stockholm, Sweden

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

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.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Irene Chiolo.

Extended data figures and tables

  1. 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, dh, k, l. P values calculated using two-tailed Mann–Whitney test. Source Data

  2. 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

  3. 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. Source Data

  4. 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. Source Data

  5. 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 ce. 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

  6. 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 df and mean ± s.d. of at least three independent experiments in a, gi. All P values calculated with two-tailed Mann–Whitney test. Source Data

  7. 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

  8. 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

  1. 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.

  2. Reporting Summary

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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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https://doi.org/10.1038/s41586-018-0242-8

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