To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin–CST–POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.
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SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination
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Custom ChaiN code is available at https://github.com/ezemiron/Chain. Custom QUANTEX code is available from the corresponding author upon reasonable request.
Numerical and statistical source data for Figs. 1e, f, 2d, e, 3a, b, e–i and Extended Data Figs. 1d, e, 2c, e, f, 3b, 4d, 5b, 6c, d, 7a, b, d, 8c, d, 9c, 10b, d are provided online. Primary imaging data underlying widefield, confocal, SIM and STED images in Figs. 1a–d, 2a–c, f, 3a–h and Extended Data Figs. 1c, i–k, 2a–c, 4b, c, e, f, h, i, 5b, 6a, c, d, 7c, e, f, 8b–g, 9b, f–h, k, l have been deposited at the European Bioinformatics Institute (EBI) BioStudies database (https://www.ebi.ac.uk/biostudies/) with accession number S-BSST275. Processed imaging data sets underlying QIBC, QUANTEX, ChaiN and other analysis, including guidance on how to navigate data sets, are available from the corresponding authors. There are no restrictions on data availability.
Lukas, J., Lukas, C. & Bartek, J. More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance. Nat. Cell Biol. 13, 1161–1169 (2011).
Setiaputra, D. & Durocher, D. Shieldin—the protector of DNA ends. EMBO Rep. 20, e47560 (2019).
Ochs, F. et al. 53BP1 fosters fidelity of homology-directed DNA repair. Nat. Struct. Mol. Biol. 23, 714–721 (2016).
Spies, J. et al. 53BP1 nuclear bodies enforce replication timing at under-replicated DNA to limit heritable DNA damage. Nat. Cell Biol. 21, 487–497 (2019).
Demmerle, J. et al. Strategic and practical guidelines for successful structured illumination microscopy. Nat. Protocols 12, 988–1010 (2017).
Kraus, F. et al. Quantitative 3D structured illumination microscopy of nuclear structures. Nat. Protocols 12, 1011–1028 (2017).
Wegel, E. et al. Imaging cellular structures in super-resolution with SIM, STED and localisation microscopy: a practical comparison. Sci. Rep. 6, 27290 (2016).
Chapman, J. R., Sossick, A. J., Boulton, S. J. & Jackson, S. P. BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. J. Cell Sci. 125, 3529–3534 (2012).
Natale, F. et al. Identification of the elementary structural units of the DNA damage response. Nat. Commun. 8, 15760 (2017).
Callen, E. et al. 53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions. Cell 153, 1266–1280 (2013).
Doil, C. et al. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 136, 435–446 (2009).
Toledo, L. I. et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 155, 1088–1103 (2013).
Bekker-Jensen, S. et al. Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J. Cell Biol. 173, 195–206 (2006).
Marnef, A. & Legube, G. Organizing DNA repair in the nucleus: DSBs hit the road. Curr. Opin. Cell Biol. 46, 1–8 (2017).
Brown, J. M. et al. A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions. Nat. Commun. 9, 3849 (2018).
Bintu, B. et al. Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 362, eaau1783 (2018).
Cardozo Gizzi, A. M. et al. Microscopy-based chromosome conformation capture enables simultaneous visualization of genome organization and transcription in intact organisms. Mol. Cell 74, 212–222.e215 (2019).
Mateo, L. J. et al. Visualizing DNA folding and RNA in embryos at single-cell resolution. Nature 568, 49–54 (2019).
Nir, G. et al. Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling. PLoS Genet. 14, e1007872 (2018).
Szabo, Q., Bantignies, F. & Cavalli, G. Principles of genome folding into topologically associating domains. Sci. Adv. 5, eaaw1668 (2019).
Densham, R. M. & Morris, J. R. The BRCA1 ubiquitin ligase function sets a new trend for remodelling in DNA repair. Nucleus 8, 116–125 (2017).
Sobhian, B. et al. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316, 1198–1202 (2007).
Miron, E. et al. Chromatin arranges in filaments of blobs with nanoscale functional zonation. Preprint at https://www.biorxiv.org/content/10.1101/566638v3 (2019).
Gupta, R. et al. DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity. Cell 173, 972–988.e923 (2018).
Hein, M. Y. et al. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163, 712–723 (2015).
Ghezraoui, H. et al. 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ. Nature 560, 122–127 (2018).
Rao, S. S. P. et al. Cohesin loss eliminates all loop domains. Cell 171, 305–320.e324 (2017).
Koch, B. et al. Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing. Nat. Protocols 13, 1465–1487 (2018).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Sartori, A. A. et al. Human CtIP promotes DNA end resection. Nature 450, 509–514 (2007).
Miron, E., Innocent, C., Heyde, S. & Schermelleh, L. In vivo and in situ replication labeling methods for super-resolution structured illumination microscopy of chromosome territories and chromatin domains. Methods Mol. Biol. 1431, 127–140 (2016).
Ball, G. et al. SIMcheck: a toolbox for successful super-resolution structured illumination microscopy. Sci. Rep. 5, 15915 (2015).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Smeets, D. et al. Three-dimensional super-resolution microscopy of the inactive X chromosome territory reveals a collapse of its active nuclear compartment harboring distinct Xist RNA foci. Epigenetics Chromatin 7, 8 (2014).
The HeLa Kyoto cell line was a gift from S. Narumiya. The U2OS cell line stably expressing RINN1/SHLD3 was a gift from C. Choudhary. The HCT116 cell line with endogenously integrated RAD21–mAID–mClover was a gift from E. Lieberman Aiden. The HeLa cell line stably expressing Histone H2B–GFP was a gift from F. Barr. The pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) plasmid was a gift from F. Zhang. The Flag-human53BP1-7A plasmid was a gift from A. Shibata. The JF585 dye was a gift from L. Lavis. Research funding for the Lukas laboratory was provided by the Novo Nordisk Foundation (grants NNF14CC0001 and NNF16CC0020906). We further acknowledge support from Wellcome Trust Strategic Awards 091911 and 107457 supporting advanced microscopy at the Micron Oxford Advanced Bioimaging Unit (L.S.), Medical Research Council award MC_UU_12009/1 (J.B. and V.B.), and the Advanced Light Microscopy Facility (ALMF) at EMBL, Heidelberg, and Abberior Instruments, Göttingen (M.L). We thank J. Dreier from the Protein Imaging Platform at Novo Nordisk Center for Protein Research for help with image analysis, and P. H. Varas from the Core Facility for Integrated Microscopy for advice on super-resolution microscopy, K. Somyajit for conceptual input to this study and all members of the Lukas laboratory for suggestions.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Yujie Sun and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Experimentally derived resolution for STED and 3D-SIM instruments using nano-bead imaging under identical conditions as for image data acquisition at the indicated excitation wavelengths. Line profile is average of three lines; dotted line shows fit of a double Gaussian distribution, where the peak-to-peak distance indicates spatial resolution. b, Western blot of GFP–53BP1 U2OS cells immunostained for 53BP1, GFP and loading controls (NUDC, tubulin). c, 3D-SIM and STED images of immunostained 53BP1-MDs in U2OS cells exposed to irradiation (1 Gy, 2 h). Images were processed identically for pixel numbers and bicubic interpolation smoothing for direct comparison. d, Diameter of a 53BP1-ND in pre- and post-replicative cells determined by full width at half maximum (FWHM, n = 75) from STED data in c. e, Centre-to-centre peak distance (n = 85) of 53BP1-NDs from STED data in c. Box plots: centre line, median; box limits, 25th and 75th centiles; whiskers, minimum and maxiumum; dots, outliers. *P = 0.0356 (d), P = 0.8587 (e; NS, not significant); two-tailed non-parametric Wilcoxon rank-sum test. Pre- or post-replicative chromatin assigned based on MCM+ or MCM– status. f, Schematic depiction of 53BP1-MD. g, Western blot of U2OS cells with endogenously tagged 53BP1–GFP immunostained for 53BP1, GFP and loading control (MCM2). h, Junction PCR showing homozygous 53BP1 tagging. i–k, 3D-SIM of 53BP1 MDs in endogenously tagged U2OS-53BP1–GFP cells (i) or U2OS cells immunostained with mouse (j) or rabbit (k) 53BP1 antibodies, exposed to irradiation (1 Gy, 2 h). Scale bars, 100 nm (a); 200 nm (c, i–k). Experiments in b, d, e, g–k were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
a, 3D-SIM of GFP–53BP1-MDs in U2OS cells exposed to irradiation (1 Gy, 2 h) and immunostained for γH2AX (PCC = 0.93, n = 300 MDs) shows high colocalization of 53BP1 and γH2AX. b, STED of a γH2AX-MD in U2OS cells treated as in a. c, 3D-SIM of three different z-planes of HeLa cells expressing histone H2B–GFP, treated with 10 ng ml–1 NCS for 2 h and immunostained for γH2AX. Nuclear DNA was visualized by DAPI. Insets are magnified γH2AX-MDs. Intensity line profiles of the three fluorophores (along the white line in the insets) show colocalization of chromatin with γH2AX-MDs. d, Western blotting of U2OS cells treated with control or XRCC4 siRNA immunostained for XRCC4 and loading marker (KAP1). e, f, Intensity line profiles of 53BP1-MDs with XRCC4 (e) and RPA (f) in cells treated as in Fig. 1b; six independent examples per condition are shown. Fluorescence intensities in c, e, f were normalized to the maximum value of each profile. Scale bars, 200 nm in a, b, insets in c and 5 μm in whole-nucleus images (c). Experiments were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
Extended Data Fig. 3 Image analysis software QUANTEX and feature comparison for maximum linear dimension.
a, QUANTEX 3D image analysis workflow to analyse spatial features of 53BP1-MDs at sites of DNA damage. Step 1: 3D-SIM images were processed and segmented for cell nuclei and 53BP1-MDs using a slice-by-slice segmentation approach. Step 2: measurements of texture, morphology and geometry features were automatically derived for all segmented structures and 3D models for visual inspection were generated. Step 3: Data analysis and statistics. For more information, see Methods. b, QUANTEX analysis of principal axis length metric of 53BP1-MDs in cells treated with control or RIF1 siRNAs. Principal axis length data were derived from the same experiments as in Fig. 1a, d and represent a parallel data analysis to the metric mean breadth in Fig. 1f; n = 60. Box plots: centre lines, medians; box limits, 25th and 75th centiles; whiskers, minimum and maximum; dots, outliers. ****P = 9.4329 × 10−6 (left), 2.3092 × 10–9 (right); two-tailed non-parametric Wilcoxon rank-sum test. The experiment was biologically replicated twice with similar results. c, Spearman’s Correlation R2 value was calculated for mean breadth and principal axis length metrics derived from control (negative class, n = 90) and RIF1 depletion (positive class, n = 87) experiments combined, in order to test association. ****P = 2.74 × 10−35; two-sided Spearman’s rank correlation coefficient method.
Extended Data Fig. 4 Disruption of ordered, circular arrangement of DSB-flanking chromatin after depletion of RIF1 or 53BP1.
a, Western blotting of U2OS cells treated with control or two RIF1 siRNAs immunostained for RIF1 and loading marker (tubulin). b, 3D-SIM of GFP–53BP1-MDs in U2OS cells transfected with RIF1 siRNA #2 and treated as in Fig. 1d. c, 3D-SIM of 53BP1-MDs in U2OS cells expressing siRNA-resistant GFP–53BP1-7A mutant and depleted for endogenous 53BP1 (#2 siRNA), exposed to irradiation (1 Gy, 2 h) (top). A schematic depiction of 53BP1-7A where glutamines in 7 SQ/TQ sites are converted to alanines (bottom). d, Distribution of circular with central interchromatin space (IC centre) versus elongated (no IC centre) 53BP1-MDs in U2OS, HeLa Kyoto, RPE1-hTERT and BJ cells (n = 130 per condition) in control or RIF1-depleted cells treated with irradiation (1 Gy, 2 h). e, 3D-SIM of immunostained 53BP1-MDs in U2OS, HeLa Kyoto, RPE1-hTERT and BJ cells after control or RIF1 depletion and irradiation exposure (1 Gy, 2 h). f, A representative 3D view of an ordered, circular arrangement of GFP–53BP1-NDs in wild-type conditions (top) and disordered, elongated shapes after RIF1 depletion (bottom). MIP, maximal intensity projection; 3D opacity view is displayed in three orientations (V1–3) indicated by coloured arrows. All 3D-SIM images in this study were routinely inspected in this way. g, Western blotting of U2OS cells treated with TP53BP1 siRNA and immunostained for 53BP1 and loading marker (NUDC). h, 3D-SIM of γH2AX-MDs in U2OS cells transfected with TP53BP1 siRNA and exposed to irradiation (1 Gy, 2 h). i, 3D-SIM of GFP–53BP1-MD in U2OS cells immunostained for γH2AX and treated as in Fig. 1d. Insets in b, c, h represent magnified single 53BP1-MDs. Scale bars, 5 μm in whole-nucleus images (b, c, h), 200 nm in e, f, i and insets (b, c, h). Experiments were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
a, Schematic depiction of live-cell 3D-SIM workflow. b, Live-SIM of a chromosome locus harbouring DNA breakage under wild-type conditions. U2OS–GFP-53BP1 cells were treated with 10 ng ml–1 NCS to induce DSBs and imaged immediately for up to 22.5 min at 2.5-min intervals. Image galleries for seven fields from four independent acquisitions are displayed. Manual classification of transition stages is colour-coded. Scale bars, 200 nm. For detailed image information see Supplementary Table 1.
Extended Data Fig. 6 Live-SIM imaging of 53BP1-MDs with the underlying chromatin and after RIF1 depletion.
a, 3D-SIM of immunostained γH2AX-MDs in control or 53BP1-depleted U2OS cells treated with irradiation (1 Gy) for the indicated times. b, Western blotting of U2OS cells expressing GFP–53BP1 and H2B–Halo-Tag immunostained for 53BP1, GFP, H2B, Halo-Tag and loading marker (MCMBP). c, Live-SIM depicting an evolving GFP–53BP1-MD at a single H2B–HaloTag-labelled chromatin locus after induction of DSBs by NCS (10 ng ml–1) for the indicated time-points. Insets are magnified 53BP1-MDs. Intensity line profiles of the two fluorophores (along the white lines in the insets) show colocalization of underlying chromatin with the 53BP1-MD. Fluorescence intensities were normalized to the maximum value of each profile. d, Additional examples of live-SIM of cells treated as in Fig. 2c. Image galleries for seven fields from four independent acquisitions are displayed. Manual classification of transition stages is colour-coded. Experiments in a–c were biologically replicated twice with similar results. Scale bars, 200 nm in a, d, and insets in c; 1 μm in large fields in c. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
Extended Data Fig. 7 Analysis of RIF1 depletion, shieldin localization, and RIF1 recruitment dynamics in the context of DSB-flanking chromatin.
a, b, QIBC of fluorescence intensities associated with γH2AX-MDs (a; n = 1,000 cells per condition) and 53BP1-MDs (b; n = 1,800 cells per condition) in control or RIF1-depleted cells treated with irradiation (1 Gy) as indicated. Box plots: centre line, median; box limits, 25th and 75th centiles; whiskers, minimum and maximum; dots, outliers. ***P = 2.0631 × 10−10, **P = 4.8803 × 10−04, NS P = 0.8651 (a, left to right); ***P = 3.887 × 10−9, NS P = 0.7172 (b, left to right); two-tailed non-parametric Wilcoxon rank-sum test. c, Confocal and STED acquisitions of immunostained 53BP1-MDs in U2OS cells treated with control or RIF1 siRNAs, exposed to irradiation (1 Gy, 2 h) and displayed as single and overlay images. d, Counts of 53BP1-NDs per 53BP1-MD quantified from STED images in c (n = 70 per condition); horizonal bar shows median, P = 0.2711 (left), 0.9566 (right); Cochran–Armitage chi-square test. e, U2OS cells expressing endogenously tagged 53BP1–GFP were treated by laser microirradiation and immunostained for γH2AX and RIF1. Stars indicate times when γH2AX, 53BP1 and RIF1 were first detected at DSBs. f, 3D-SIM of 53BP1-MD and 3×-Flag-SHLD3 in U2OS cells exposed to irradiation (1 Gy, 2h) and immunostained for 53BP1 and Flag-tag (six independent examples are shown). Scale bars, 200 nm (c, f), 20 μm (e). Experiments were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
Extended Data Fig. 8 RASER-FISH analysis of 53BP1-MDs at site-specific DSBs in KIF23 and KIF11 loci.
a, Depiction of a 0.45-Mb TAD from a reference cell line (adapted from Yue laboratory 3D genome browser, see Methods) harbouring the KIF23 gene (top) and a 0.4-Mb TAD harbouring the KIF11 gene (bottom). Sites of CRISPR–Cas9 site-specific DSBs and a position of each RASER-FISH probe are indicated. b, DAPI-stained U2OS cells transfected with Cas9 ribonucleoprotein complexes with control, KIF23-, or KIF11-targeting guide RNAs (gRNA). Arrows indicate examples of mitotic aberrations inflicted by KIF23 and KIF11 knockout. c, d, 3D-SIM of the KIF23-TAD (c) and the KIF11-TAD (d) RASER-FISH probes in cells treated as in Fig. 3a, b but at loci without DNA damage (no 53BP1 signal). Dual-colour FISH probes FP-A and FP-B are located within the same TAD in c; FP-C and FP-D in in two adjacent TADs in d. e, Widefield microscopy of immunostained 53BP1-MDs at the damaged KIF23-TAD locus labelled by FP-B in U2OS and RPE1-hTERT cells 3 h after transfection with KIF23 gRNA–Cas9. Insets (MD1–3) are magnified 53BP1-MDs shown in xy, xz and yz orientations. f, Widefield microscopy of immunostained 53BP1-MDs at the damaged KIF11-TAD locus labelled by FP-C in U2OS cells 3 h after transfection with KIF11 gRNA–Cas9. Insets (MD1–3) were generated as in e. g, 3D-isosurface projections (V1–3) of 3D-SIM images of FP-C- and FP-D-labelled KIF11 TADs after DNA damage induction as shown in Fig. 3b. Scale bars, 5 μm in whole-nucleus images (e, f), 200 nm in insets (e, f) and in c, d, 20 μm in b. Experiments in b–f were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1.
Extended Data Fig. 9 Disruption of ordered, circular arrangement of DSB-flanking chromatin after cohesin depletion.
a, Western blotting of HCT116-RAD21–mAID–mClover cells treated with auxin (aux) as indicated and immunostained for RAD21 and loading marker (NUDC). b, Widefield images of HCT116-RAD21–mAID–mClover cells, either untreated or treated with auxin for 6 h to induce RAD21 degradation. c, QUANTEX analysis of mean breadth of 53BP1-MDs in cells treated as in Fig. 3c, d (n = 110). Box plots: centre line, median; box limits, 25th and 75th centiles; whiskers, minimum and maximum; dots, outliers. ****P = 3.8495 × 10−17 for MCM+, ****P = 7.636 × 10−16 for MCM–; two-tailed non-parametric Wilcoxon rank-sum test. d, Western blotting of U2OS cells treated with control or RAD21 siRNA, immunostained for RAD21 and loading marker (tubulin). e, Western blotting of U2OS cells treated with control or SMC1 siRNA, immunostained for SMC1 and loading marker (MCMBP). f–h, 3D-SIM of GFP–53BP1-MDs in U2OS cells transfected with RAD21 siRNA (f), SMC1 siRNA #1 (g), or SMC1 siRNA #2 (h) and exposed to irradiation (1 Gy, 2 h). i, Western blotting of U2OS cells treated with the indicated siRNAs and immunostained for γH2AX; total protein stain is loading control. j, Western blotting of U2OS cells treated with indicated siRNAs and immunostained for 53BP1 and loading marker (MCM7). k, l, 3D-SIM of GFP–53BP1-MDs in U2OS cells treated with 10 μM DNA-PK inhibitor (k) or RBBP8 (also known as CtIP) siRNA (l) and exposed to irradiation (1 Gy, 2 h). m, Western blotting of U2OS cells treated with control or RBBP8 siRNA, immunostained for CtIP and loading marker (NUDC). Insets in (f–h, k, l) are magnified 53BP1-MDs. Scale bars, 5 μm in whole nuclei (f–h, k, l), 200 nm in insets (f–h, k, l), 20 μm in b. Experiments were biologically replicated twice with similar results. For detailed image information see Supplementary Table 1. For gel source data see Supplementary Fig. 1.
Extended Data Fig. 10 Chromatin density analysis by ChaiN, RNA-seq data, and a schematic model for topological surveillance of DSB loci.
a, Schematic depiction of ChaiN analysis to quantify chromatin density in 3D-SIM images based on histone H2B–GFP distribution. Reconstructed and aligned 3D-SIM images were used to segment volumes occupied by 53BP1-MDs and subjected to an HMM process to derive seven discrete GFP–H2B chromatin density classes within the segmented region. Class 1 represents chromatin-free interchromatin space, while class 2–7 feature increasing chromatin densities. An equivalent analysis of the whole nucleus serves as a control for global chromatin distributions outside 53BP1-MDs. b, ChaiN analysis in undamaged nuclei in wild-type or RIF1-depleted cells (n = 12 per condition). Median ± 95% CI. *P = 0.0348, 0.0226 (class 2 and 4), NS P = 0.2525, 0.7373, 0.0990, 0.4874, 0.9496 (classes 1, 3, 5–7); two-tailed Student’s t-test. c, A hypothetical model. A DSB triggers accumulation of 53BP1 in the damaged and several neighbouring chromatin nanodomains. Saturation of 53BP1 at chromatin nanodomains prompts recruitment of RIF1 to the boundaries between them. Through functional crosstalk with cohesin, RIF1 locally stabilizes the nanodomain topology into an ordered and circular microdomain, which confines repair factors such as BRCA1 to DSBs and locally concentrates shieldin–CST–POLα to restrain DNA-end resection. Absence of RIF1 leads to topological disorder that results in excessive spreading of BRCA1, inability to concentrate DNA-end protection factors and DSB hyper-resection. d, RNA-seq data for TP53BP1, RIF1 and SHLD1 transcripts per million kilobases in cancerous cells (U2OS, HeLa) and normal cells (IMR90, HBL100). Data were derived from publicly available RNA-seq data in the EMBL-EBI expression atlas (see Methods). Scale bars (a), 5 μm in the whole nucleus and 200 nm in the magnified 53BP1-MD image (right). For detailed image information see Supplementary Table 1.
This figure contains all uncropped versions of the cropped western blots shown in the Extended Data Figures. Black boxes indicate cropped regions. Asterisk indicates unspecific protein band.
Methods, equipment and settings for microscopy. This table details information on imaging techniques, microscope equipment, image display, image processing, fluorophores and pseudo-colouring and sample preparation for all images shown in the main and extended data figures.
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Ochs, F., Karemore, G., Miron, E. et al. Stabilization of chromatin topology safeguards genome integrity. Nature 574, 571–574 (2019). https://doi.org/10.1038/s41586-019-1659-4
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