Volume 63, Issue 2, Pages 293-305 (July 2016) Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin  Katerina Tsouroula, Audrey Furst, Melanie Rogier, Vincent Heyer, Anne Maglott-Roth, Alexia Ferrand, Bernardo Reina-San-Martin, Evi Soutoglou  Molecular Cell  Volume 63, Issue 2, Pages 293-305 (July 2016) DOI: 10.1016/j.molcel.2016.06.002 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Cas9-Specific Induction of DSBs at Pericentric Heterochromatin (A) Expression of Cas9-EGFP with a major satellite-specific gRNA in mouse NIH 3T3 cells generates DSBs in pericentric heterochromatin (DAPI-dense regions). (B) Immunofluorescence (IF) confocal analysis of cells expressing Cas9-EGFP or dCas9-EGFP ± gRNA and stained with DAPI and antibodies specific for γ-H2AX and 53BP1. (C) IF confocal analysis of cells expressing Cas9-EGFP ± gRNA and stained with DAPI and antibodies specific for 53BP1 and pATMS1981 (top) or γ-H2AX and MDC1 (bottom). (D) IF confocal analysis of cells expressing Cas9-EGFP+gRNA after treatment with ATM (ATMi) or ATR (ATRi) inhibitor or vehicle (DMSO) and stained with DAPI and a γ-H2AX-specific antibody. (E) Western blot analysis for Cas9-EGFP (EGFP), γ-H2AX, pATMS1981, pKAP1S824, KAP1, pChk1S345 and tubulin in protein extracts prepared from cells expressing Cas9-EGFP ± gRNA 8 or 16 hr post-transfection. As a comparison, NIH 3T3 cells were treated with increasing concentrations of NCS. Theoretical molecular weights are indicated. For confocal images, scale bars represent 10 μm. See also Figure S1. Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Cell-Cycle-Specific Regulation of DSB Localization in Pericentric Heterochromatin (A) Super-resolution imaging of cells expressing Cas9-EGFP+gRNA and stained with DAPI and antibodies specific for γ-H2AX (top; Movie S1) or 53BP1 (bottom; Movie S2). Quantification is shown on the right. (B) Super-resolution imaging analysis of G2 (RO-3306-treated) cells expressing Cas9-EGFP+gRNA and stained with DAPI and antibodies specific for γ-H2AX (top; Movie S3) or 53BP1 (bottom; Movie S4). Quantification is shown on the right. (C) Quantification of γ-H2AX pattern in G1 (EdU−/H3S10p−) and G2 (RO-3306-treated, EdU−/H3S10p+) cells stably expressing Cas9 and transfected with in vitro transcribed major satellite-specific gRNA for the indicated time points and stained with DAPI and antibody specific for γ-H2AX. For super-resolution images, scale bars represent 5 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S2 and S3. Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Spatial and Temporal Separation of NHEJ and HR Factor Recruitment in Heterochromatic Structures (A and B) IF confocal analysis of cells expressing Cas9-EGFP+gRNA specific for major satellite DNA repeats in G1 and G2 and stained with DAPI and antibodies specific for (A) γ-H2AX and RAD51 or (B) γ-H2AX and Ku80. Quantification is shown on the right. (C and D) IF confocal analysis of cells expressing Cas9-EGFP+gRNA specific for minor satellite DNA repeats in G1 and G2, stained with DAPI and antibodies specific for (C) γ-H2AX and RAD51 or (D) γ-H2AX and Ku80. Quantification of RAD51 or Ku80 recruitment is shown on the right. Scale bars represent 10 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S4 and S5. Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Cas9-Induced DSBs Induce Chromatin Expansion Independently of the Release of HP1s or Associated Modifications (A–I) Schematic representation of the high-throughput analysis (see Supplemental Experimental Procedures) to measure the intensity of γ-H2AX (B), pKAP1S824 (C), HP1α (E), HP1β (F), HP1γ (G), H3K9me3 (H), and KAP1 (I) and the chromocenter (DAPI-dense) area (D). For all plots, individual cell values are represented as box-and-whisker plots (median and quartiles with outliers representing 1% of the population) of two independent experiments with n > 300 cells for G1 and n > 350 cells for G2 (RO-3306 treated cells). Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Compacted Chromatin Is Not Refractory to the Recruitment of RAD51 (A–C) Quantification of γ-H2AX pattern (A), RAD51 (B), or Ku80 (C) in G1 and G2 cells expressing Cas9-EGFP+gRNA and treated with DMSO or TSA, expressing Cas9-EGFP+gRNA or Cas9-EGFP-VP64+gRNA or depleted for KAP1, HP1α, HP1β, and HP1γ. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figure S6. Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 DSB Relocation at the Periphery of the Heterochromatin Domain Is Dependent on DNA End Resection (A) IF confocal analysis of G2 cells expressing Cas9-EGFP+gRNA stained with DAPI and antibodies specific for 53BP1 and RPA32. (B–E) Quantification of γ-H2AX pattern in G2 cells expressing Cas9-EGFP+gRNA after treatment with DMSO or Mirin inhibitor (B), CtIP knockdown (C), GAM-EmGFP expression (D), or Cas9-CtIP expression (E). (D) IF confocal analysis of G2 cells expressing Cas9-mCherry+gRNA + GAM-EmGFP stained with DAPI and a γ-H2AX-specific antibody is shown. Scale bars represent 10 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S7A–S7G. Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 The RAD51/BRCA2 Complex Stabilizes DSBs at the Periphery of Heterochromatin (A and B) Quantification of RAD51 recruitment (A) and γ-H2AX pattern (B) in G2 cells expressing Cas9-EGFP+gRNA under BRCA2 knockdown conditions. (C) Quantification of γ-H2AX pattern in G2 cells expressing Cas9-RAD51 (left) or Cas9-BRC3 or Cas9-mBRC3 (right). (D) IF confocal analysis of G2 cells expressing Cas9-RAD51 or Cas9-BRC3+gRNA stained with DAPI and specific antibodies for γ-H2AX and RAD51. (E) IF confocal analysis of G1 and G2 cells expressing Cas9-mCherry + gRNA + RAD52-EGFP stained with DAPI and specific antibodies for γ-H2AX and RAD51. (F) Quantification of RAD52 recruitment and pattern in G1 and G2 expressing Cas9-mCherry + gRNA + RAD52-EGFP. (G) Quantification of γ-H2AX pattern in G2 cells expressing Cas9-EGFP + gRNA under RAD52 knockdown conditions. Scale bars represent 10 μm. Values represent mean ± SD of three independent experiments with n = 50 cells. See also Figures S7H–S7M. Molecular Cell 2016 63, 293-305DOI: (10.1016/j.molcel.2016.06.002) Copyright © 2016 Elsevier Inc. Terms and Conditions