Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, ‘safe’ homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the heterochromatin domain before strand invasion. Here we show that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in heterochromatin, and identifies the nuclear periphery as a specialized site for heterochromatin repair in a multicellular eukaryote.
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This work was supported by the USC Gold Family Fellowship and the USC Research Enhancement Fellowship to T.R.; the USC Provost Fellowship to B.S.; R21ES021541, The Rose Hills Foundation, and R01GM117376 to I.C.; R01GM086613 to G.H.K. We would like to thank S. Keagy, M. Michael, J. Haber and O. Aparicio for insightful comments on the manuscript, and S. Gasser for sharing results before publication. We are grateful to V. Doye (Institut Jacques Monod, France), J. Kadonaga (University of California San Diego, USA), J. Fischer (University of Texas, USA), M. Welte (University of Rochester, USA), A. Orian (Technion, Israel), S. Parkhurst (Fred Hutchinson Cancer Research Center, USA), A. Ashworth (Institute of Cancer Research, UK) and the O. Aparicio laboratory (University of Southern California, USA) for sharing reagents and the Chiolo and Karpen laboratories for helpful discussions. We thank C. Ferraro and N. Brisson for their help with Lamin and SUMO RNAi studies, and J. Swenson for his initial dPIAS RNAi studies. We also thank M. Bonner for generating the mCh–LaminC construct, D. Das, E. Lin and C. Ren for cloning and RNAi reagents, A. Kim, S. Wijekularatne and N. Saxena for cloning and Smc5 mutant characterization. Fly stocks from BDSC (NIH P40OD018537) and RNAi libraries from DRSC (NIH R01GM067761) were used for this study.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Smc5/6 and dPIAS are independently recruited to heterochromatic DSBs and do not affect euchromatic repair kinetics.
(a) IF of GFP-Nse2-expressing cells 30 min after IR, stained for GFP and γH2Av. (b) Images of cells expressing mCh-HP1a and one of the GFP-tagged Smc5/6 subunits, before (−) and 15 min after (+) IR. (c) Images (left) and quantitation (right) of Nse2 foci in GFP-Nse2/mCherry-HP1-expressing cells 15 min after IR, after RNAi depletions (∗∗∗∗p < 0.0001, n > 130 cells. The error bars represent mean ± s.d. derived from three independent experiments, whereas the sample size used to determine P values is the total number of cells for each RNAi, pooled across the three experiments). (d) Quantitation of the experiment described in Fig. 1c shows the frequency of foci where dPIAS is brighter (>), of similar intensity (=), or dimmer (<) than γH2Av before and after IR. Error bars: s.e.m., n > 20 cells/timepoint from one experiment. (e) Western analysis shows Smc5/6 RNAi efficiency (actin: loading control). (f) IF (left) of the experiment described in Fig. 1d shows γH2Av foci in DAPI-bright after RNAi depletions. Quantitation (right) shows the total numbers of γH2Av foci. (g) Western analysis of FHA-Nse2-expressing cells shows Nse2 RNAi efficiency (tubulin: loading control). (h) qPCR analysis shows dPIAS RNAi efficiency. (i) IF (left) and quantitation (right) show SUMO RNAi efficiency. (j) Images (top) and quantitation (bottom) of cells expressing mC-HP1a plus GFP-Nse2 or GFP-Smc6, show the effect of RNAi depletions on Nse2/Smc6 recruitment to the heterochromatin domain before IR, and repair foci 10 min after IR. Smc6 foci require Nse4/Smc5, indicating the presence of the core complex at DSBs. Bottom right: Quantitation of Kc cells fixed 10 min after IR and processed as in Fig. 1c, shows RNAi effects on dPIAS recruitment to γH2Av foci. (Error bars: s.e.m., ∗∗∗∗p < 0.0001, n > 16 cells/RNAi from one experiment). (k) As described in f, except Rad51 foci were analyzed. Images (left) and quantitation (right) correspond to the experiment described in Fig. 1e. (l) Western analysis shows Rad51 RNAi efficiency (actin: loading control). Ctrl/C = control. Scale bars = 1 μm. Images are projections of all Z-stacks (a–c,j,i) or Z-stacks spanning the DAPI-bright region (f,k). Exact n values are in Supplementary Table 1.
Supplementary Figure 2 STUbL and RENi do not affect Smc5/6 association with heterochromatin and repair foci, and euchromatic repair.
(a) Western analysis of cells expressing GFP-Dgrn shows Dgrn RNAi efficiency, and that dRad60 RNAi does not affect Dgrn protein level. (∗) A non-specific band is the loading control. (b) Western analysis of cells expressing FHA-dRad60 shows dRad60 RNAi efficiency, and that Dgrn RNAi does not affect dRad60 protein level. Actin is the loading control. (c) Quantitation of the experiment described in Fig. 2b shows the number of γH2Av foci in DAPI-weak after RNAi depletions. (d) As in Fig. 2b except that 2.5 or 10 Gy were used to induce DSBs (p < 0.05 for all comparisons versus Ctrl at either 2.5 Gy or 10 Gy, n > 120 cells; error bars represent mean ± s.d. from three independent experiments, whereas the sample size used to determine P values is the total number of cells for each RNAi and dose, pooled across the three experiments). (e) Images (left) and quantitation (right) of Nse2 or Smc6 signals in cells described in Supplementary Fig. 1j show the effects of dRad60 or Dgrn RNAi on Nse2/Smc6 colocalization with the HP1a domain before IR, or Nse2/Smc6 recruitment to damage foci 10 min after IR. Images are maximum intensity projections of nuclei. Scale bars = 1 μm. Error bars: s.e.m.; n = 17 cells/RNAi condition from one experiment. (f) qPCR analysis shows dRad60 RNAi efficiency in the experiment described in g. (g) Ip of GFP-Dgrn was done as in Fig. 2d, except that dRad60 or a control was depleted before Ip. (h) Quantitation of the experiment described in Fig. 2e shows the number of foci in DAPI-weak. Because the number of Rad51 foci in DAPI-bright is a small fraction of the total number of foci, the number of foci in DAPI-weak is similar to the total number of foci (not shown). Ctrl/C = control.
Supplementary Figure 3 Nuclear pores and INMPs are required for relocalizing heterochromatic DSBs and do not affect euchromatic repair kinetics.
(a) Top: western blots show Nup153 and Mtor RNAi efficiencies. Lamin and a non-specific band (∗) are loading controls. Middle: IF analyses and quantitations show Nup107 or Koi RNAi efficiencies. Bottom: qPCR analyses show Nup160, Nup205, Nup93, Nup50, or Spag4 RNAi efficiencies. (b) Top: IF analyses show the effect of Nup153, Nup107 or Nup160 RNAi depletion on nuclear pore stability (FG-porin signal, labeled with MAb414 antibody) and Nup107 association with the nuclear periphery. Nup153 RNAi effects were independently confirmed with a second set of siRNAs (not shown). Bottom: IF analysis shows no effects of Mtor, Nup50, Nup205 or Nup93 RNAi on Nup107 recruitment to the nuclear periphery. (c) Quantitation of cells fixed before and after IR and processed for IF shows the number of γH2Av foci in DAPI-bright (left) and DAPI-weak (right) after RNAi depletion of the indicated proteins (error bars: s.e.m., ∗∗∗∗p < 0.0001 for Nup153 versus Ctrl RNAi, two-tailed Mann-Whitney test with n > 87 cells for either 60 min or 4 h and for each RNAi condition, from one experiment). Note: Ctrl RNAi samples are the same as in Fig. 3b, d. (d–h) Quantitations of DAPI-weak foci in the experiments described in Fig. 3b (related to Supplementary Fig. 3d), Fig. 3c (related to Supplementary Fig. 3e), Fig. 3d (related to Supplementary Fig. 3f), Fig. 3e (related to Supplementary Fig. 3g), and Fig. 3f (related to Supplementary Fig. 3h). (i) As in Fig. 3f except that 2.5 or 10 Gy were used to induced DSBs (p < 0.05 for all comparisons versus Ctrl at either 2.5 Gy or 10 Gy, n > 140 cells; error bars represent mean ± s.d. from three independent experiments, whereas the sample size used to determine P values is the total number of cells for each RNAi and dose, pooled across the three experiments). Ctrl/C = control. Scale bars = 1 μm. Images are middle Z-stacks of nuclei. Exact n values are in Supplementary Table 1.
Supplementary Figure 4 Nuclear pores and INMPs recruit STUbL/RENi to the nuclear periphery, and do not prevent aberrant recombination in heterochromatin.
(a) Quantitation of the experiment described in Fig. 4a shows the frequency of cells with a ‘clustering’ phenotype after Lamin RNAi, relative to cells with intact Lamin signal (normal) and cells without Lamin (no Lamin). (b) IF analysis of cells expressing GFP-Spag4, fixed after Lamin RNAi and stained for Nup153 (nuclear pore marker), Koi and GFP. (c) Western analyses of cells expressing GFP-Dgrn and FHA-dRad60 show the levels of Dgrn (left) or dRad60 (right) proteins after Nup107+Koi+Spag4 RNAi relative to Ctrl RNAi. Actin and tubulin are loading controls. (d) IF analysis of cells expressing FHA-dRad60 and GFP-Dgrn with anti-GFP (top) or-HA (bottom) antibodies, after RNAi depletion of the indicated proteins. (e) IF analysis of cells expressing FHA-dRad60 and GFP-Dgrn from the experiment described in Fig. 4c were stained for Nup153, Koi, and GFP (left) or HA (right). (f) Cells were fixed 60 min post IR after RNAi depletions, and analyzed by IF to detect Rad51 foci. Quantifications show the frequencies of foci relative to DAPI-bright and DAPI-weak regions (p < 0.0001 for Smc5/6 RNAi versus Ctrl RNAi in DAPI-bright, n > 160 cells; error bars represent mean ± s.d. from three independent experiments, whereas the sample size is the total number of cells for each RNAi, pooled across the three experiments). (g) Quantitation of DNA filaments connecting dividing cells in cells fixed and processed as in Fig. 1f (n > 700 cells, error bars: mean ± s.d. from three independent experiments). Smc5 and Ctrl RNAi samples are the same as in Fig. 1f. (h) Quantitation of foci in DAPI-weak for the experiment described in Fig. 4d. Ctrl/C = control. Scale bars = 1 μm. Images are middle Z-stacks of nuclei. Exact n values are in Supplementary Table 1.
Supplementary Figure 5 Relocalization of repair foci to the nuclear periphery correlates with Rad51 recruitment.
(a) Quantitation of the experiment described in Fig. 5a shows the total number of γH2Av foci detected in the middle Z-stack or in the entire nucleus (all Z stacks). (b) IF analysis (top) and quantitation (bottom) of cells expressing mCh-HP1a show HP1a RNAi efficiency. (c) Quantitation of the experiment described in Fig. 5b shows the total number of γH2Av foci after RNAi depletions of the indicated components. Only middle Z-stacks of nuclei were used for this analysis. (d) IF analysis of cells expressing GFP-Mu2, fixed 60 min after IR, and stained for GFP, Rad51 and Koi (nuclear periphery), shows colocalizations between Mu2 and Rad51 at the nuclear periphery (highlighted in the zoomed detail). Ctrl = control. Scale bars = 1 μm. Images are maximum intensity projections (b) or middle Z-stacks (d) of nuclei.
Supplementary Figure 6 Relocalization is required for heterochromatin repair and stability.
(a) Image of a cell expressing GFP-ATRIP, mCh-HP1a and mCh-LaminC at 10 min after IR shows that HP1a and LaminC signals are distinguishable from each other. This cell line was used in Fig. 6a. (b) Quantitation of the experiment described in Fig. 6b shows the kinetic of disappearance of γH2Av foci outside the H3K9me2 domain (‘euchromatic space’), after RNAi depletions. (c) Cells from the 20 h timepoint of the Nup107+Koi+Spag4 RNAi experiment described in Fig. 6b and (b) were stained for TUNEL, and IF was performed to detect H3K9me2 and γH2Av. Image analysis (left) and quantitation (right) show that about 75% of γH2Av foci associated with H3K9me2 (+), and 5% of those outside the H3K9me2 domain (−), overlap with TUNEL signals. Error bars: s.e.m., n = 74 foci from one experiment. (d) IF analysis (left) and quantitation (right) show the frequency of apoptotic cells (Caspase3-positive) after RNAi depletions. Data were normalized to corresponding non-irradiated samples (p < 0.0001 for all depletions versus Ctrl RNAi, n > 1100 cells; error bars represent mean ± s.d. from three independent experiments, whereas the sample size is the total number of cells for each RNAi, pooled across the three experiments). (e) Quantitation of non-irradiated (-IR) cells from the experiment described in Fig. 6d. (f) Western analyses of protein extracts from yry, smc57/7 (smc57), smc519/19 (smc519)and Smc517/17 (Smc517) flies show Smc5 protein levels relative to control flies. Tubulin is the loading control. (g) Quantitations of the experiment described in Fig. 6e show that most aneuploidies (left) correspond to the loss of 4th or Y chromosomes, or the loss of whole arms of other chromosomes. Most chromosome fusions (right) involved 4th or Y chromosomes or occurred at centromeric/pericentromeric regions. Error bars: s.e.m., n > 40 karyotypes for each genotype. Ctrl = control. Scale bars = 1 μm in a,c and 5 μm in d. Images are middle Z stacks (a), maximum intensity projections spanning the heterochromatin domain (c), or maximum intensity projections of entire nuclei (d). Exact n values are in Supplementary Table 1.
Supplementary Figure 7 Uncropped scans of the gels shown in Fig. 2c, d.
Dashed boxes indicate the portion of each gel included in the figure. (∗) indicates Smc6 background signals from a previous hybridization. (∗∗) indicates a non-specific band in Kc cells.
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Ryu, T., Spatola, B., Delabaere, L. et al. Heterochromatic breaks move to the nuclear periphery to continue recombinational repair. Nat Cell Biol 17, 1401–1411 (2015). https://doi.org/10.1038/ncb3258
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