Volume 47, Issue 3, Pages 383-395 (August 2012) Tandem Protein Interaction Modules Organize the Ubiquitin-Dependent Response to DNA Double-Strand Breaks  Stephanie Panier, Yosuke Ichijima, Amélie Fradet-Turcotte, Charles C.Y. Leung, Lilia Kaustov, Cheryl H. Arrowsmith, Daniel Durocher  Molecular Cell  Volume 47, Issue 3, Pages 383-395 (August 2012) DOI: 10.1016/j.molcel.2012.05.045 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 RNF169 Is a RNF168 Paralog (A) Domain structure of RNF168 and RNF169. Amino acid positions and domains are indicated. (B and C) U2OS cells transfected with the indicated siRNAs were irradiated and processed for RNF169 and γ-H2AX immunofluorescence. Scale bars represent 8 μm. (D) Quantitation of (B) and (C). At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (E) Flp-In/T-Rex HCT116 cell lines expressing the indicated RNF169 mutants were irradiated and processed for Flag and γ-H2AX immunofluorescence. The percentage of cells in the population that display the represented phenotype is indicated on each micrograph. The scale bar represents 8 μm. (F) Pull-down assay using MBP-RNF168 and MBP-RNF169 incubated with either Lys48- or Lys63-linked Ub2–7 chains (ubK48 or ubK63, respectively) and analyzed by immunoblotting (IB). See also Figure S1. Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Identification of the LRM2 in RNF168 and RNF169 (A) U2OS cells transfected with the indicated GFP-tagged RNF168 constructs were irradiated (10 Gy) and processed for GFP fluorescence. At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (B) U2OS cells were first transfected with the indicated siRNAs. Twenty-four hours later, cells were transfected with siRNA-resistant Flag-tagged RNF168 constructs (RNF168∗). Twenty-four hours after DNA transfection, cells were irradiated (10 Gy) and processed for Flag immunofluorescence. At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (C) Model of RNF168 recruitment to DSBs. (D) U2OS cells transfected with the indicated GFP-tagged RNF169 constructs were processed for GFP immunofluorescence as in (A). At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (E) Alignment of RNF168 and RNF169 homologs. Conserved residues are shaded. Arrows indicate RNF169 residues mutated in (F). (F–H) U2OS cells transfected with the indicated GFP-tagged RNF169 (F and G) or RNF168 (H) constructs were processed for GFP and γ-H2AX immunofluorescence as in (A). Scale bars represent 7 μm. At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (G) The percentage of cells in the population that display the represented phenotype is indicated on each micrograph. (I) Pull-down assay with MBP-RNF168 (WT or L476A/R477A [LARA] mutant) and MBP-RNF169 (WT or L699A/R700A [LARA] mutant) incubated with Lys63-linked Ub2–7 chains (ubK63) and analyzed by immunoblotting (IB). See also Figures S2 and S3. Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 RNF8 Ubiquitylation Products Are Recognized by the N Terminus of RNF168 (A) U2OS cells were first transfected with the indicated siRNAs. Twenty-four hours later, cells were transfected with the indicated GFP-tagged RNF168 and RNF169 constructs (∗ denotes siRNA-resistance). Twenty-four hours after DNA transfection, cells were irradiated and processed for GFP and γ-H2AX immunofluorescence. The scale bar represents 7 μm. (B) Quantitation of (A). At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (C) Pull-down assay using MBP-RNF168 and -RNF169 proteins that were incubated with Lys63-linked Ub2–7 chains (ubK63) and analyzed by immunoblotting (IB). Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Mapping of LRM1 (A) U2OS cells were first transfected with the indicated siRNAs. Twenty-four hours later, cells were transfected with the indicated siRNA-resistant GFP-tagged RNF168 constructs. Twenty-four hours after DNA transfection, cells were irradiated (10 Gy) and processed for GFP and γ-H2AX immunofluorescence. At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (B) Pull-down assay using MBP-RNF168 proteins that were incubated with Lys63-linked Ub2–7 chains (ubK63) and analyzed by immunoblotting (IB). (C) Schematic of the LacO/LacR chromatin-targeting system. (D) U2OS LacO SceI 111 II cells were transfected with the indicated mCherry-LacR-RNF8 (LacR-RNF8) constructs and processed for mCherry and 53BP1 or RNF168 immunofluorescence. The scale bar represents 7 μm. (E) U2OS LacO SceI 111 II cells were cotransfected with mCherry-LacR-RNF8 and the indicated GFP-tagged RNF168 constructs and processed for mCherry and GFP immunofluorescence. The scale bar represents 7 μm. (F) Quantification of (E). GFP-RNF168 relative mean fluorescence intensity (RMFI) at LacR-RNF8 arrays was quantified with ImageJ software (National Institutes of Health [NIH]). At least 40 arrays were analyzed. Data are depicted relative to cells expressing GFP only and are represented as the mean ± SD (n = 3). See also Figure S4. Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 The LRM2 Is Transferable and Binds Nucleosomes (A and B) U2OS LacO SceI 111 II cells were cotransfected with mCherry-LacR-RNF168 (LacR-RNF168) and the indicated GFP-tagged UBD-LRM2 constructs and processed for mCherry and GFP immunofluorescence. Representative micrographs are shown in the left panel, and quantitation is shown in the right panel. Scale bars represent 7 μm. GFP-UBD-LRM2 relative mean fluorescence intensity (RMFI) at LacR-RNF168 arrays was quantified with ImageJ software (NIH). At least 40 arrays were analyzed. Data are depicted relative to cells expressing GFP only and are represented as the mean ± SD (n = 3). (C) Whole-cell extracts of U2OS LacO SceI 111 II cells expressing the indicated GFP-tagged UBD-LRM constructs were separated by SDS-PAGE and analyzed by immunoblotting (IB). Tubulin was used as loading control. (D) Peptide pull-downs of HEK293T chromatin extracts with biotinylated RNF169 LRM2 peptides (WT or L699A/R700A [LARA] mutant). The pull-down in the LARA 2X lane was carried out with twice the amount of peptide. The pull-downs were analyzed by immunoblotting (IB). (E) Recombinant nucleosomes (Nuc) were separated by SDS-PAGE and visualized by Coomassie brilliant blue (CBB) staining. (F) Peptide pull-downs of recombinant nucleosomes with biotinylated RNF169 LRM2 peptides (WT or L699A/R700A [LARA] and R689A mutants) or a scrambled control. The pull-downs were analyzed by immunoblotting (IB). Note that H2A input and corresponding peptide pull-down lanes were cropped from the same film. For consistency, the H2A input was moved from the right side to the left side of the figure. (G) Chromatin isolated from HEK293 cells overexpressing RNF168 was subjected to pull-downs with MBP (CTRL), MBP-RNF169662–708 (WT), and the A673G or L699A/R700A (LARA) mutants. Proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. See also Figure S5. Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 RNF169 Inhibits 53BP1 IR-Induced Foci (A) U2OS cells transfected with the indicated Flag-tagged RNF169 constructs were irradiated and processed for Flag and 53BP1 immunofluorescence. The scale bar represents 7 μm. (B) Quantitation of (A). At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (C) U2OS cells transfected with the indicated siRNAs were irradiated and processed for 53BP1 immunofluorescence. DNA was counterstained with DAPI. The scale bar represents 7 μm. (D) Quantitation of (C). At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). See also Figure S6. Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 Identification of LRM Motifs in RAD18 and RAP80 (A) Pull-down assay using MBP-RAD18 proteins that were incubated with Lys63-linked Ub2–7 chains (ubK63) and analyzed by immunoblotting (IB). (B) Top: Domain structure of RAD18. Amino acid positions and domains are indicated. Bottom: U2OS cells transfected with the indicated GFP-tagged RAD18 constructs were irradiated (10 Gy) and processed for GFP fluorescence. At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (C) Whole cell extracts of U2OS cells expressing the indicated GFP-tagged RAD18 constructs were separated by SDS-PAGE and analyzed by immunoblotting (IB). Tubulin was used as loading control. (D) Top: Domain structure of RAP80. Amino acid positions and domains (AIR, Abraxas-interacting region; ZnF, zinc finger) are indicated. Bottom: U2OS cells transfected with the indicated GFP-tagged RAP80 constructs were irradiated (10 Gy) and processed for GFP fluorescence. At least 100 cells per condition were counted. Data are represented as the mean ± SD (n = 3). (E) Whole-cell extracts of U2OS cells expressing the indicated GFP-tagged RAP80 constructs were separated by SDS-PAGE and analyzed as in (C). (F) U2OS cells transfected with the indicated Flag-tagged RAP80 constructs were irradiated and processed for Flag and γ-H2AX immunofluorescence. The percentage of cells in the population that display the represented phenotype is indicated on each micrograph. The scale bar represents 7 μm. See also Figure S7. Molecular Cell 2012 47, 383-395DOI: (10.1016/j.molcel.2012.05.045) Copyright © 2012 Elsevier Inc. Terms and Conditions