Molecular Cell, Volume 47

Supplemental Information

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, and Daniel Durocher

SUPPLEMENTAL EXPERIMENTAL PROCEDURES Plasmids and stable cell lines To generate RNF168 expression vectors, the open reading frame of RNF168 was PCR-amplified from the siRNA-resistant human RNF168 cDNA described in (Stewart et al., 2009) and inserted into pcDNA3-GFP-NLS, pMALC2- His6 or the mCherry-lacR-NLS vector (a gift from E. Soutoglou). To generate RNF169 expression vectors, the cDNA for human RNF169 (obtained from Kazusa, Japan; pF1KSDA1991) was amplified by PCR and ligated into pcDNA3-GFP-NLS, pMALC2- His6 or pcDNA5-FRT-TO-flag. To generate the mCherry-LacR-RNF8 expression vector, RNF8 was PCR-amplified from the human RNF8 cDNA described in (Kolas et al., 2007) and ligated into the mCherry-lacR-NLS vector. The cDNAs of the UBD-LRM2 chimeras (MIURx5-LRM2 and UBAHR23A-LRM2) were synthesized by GeneArt (Invitrogen) and ligated into pcDNA3-GFP-NLS. To generate RAD18 expression vectors, the cDNA for human RAD18 (clone V54067 from Openfreezer) was amplified by PCR and ligated into pcDNA3-GFP-NLS and pMALC2- His6. To generate RAP80 expression vectors, the cDNA for human RAP80 (obtained from Openfreezer) was amplified by PCR and ligated into pcDNA3-GFP-NLS and pcDNA5-FRT-TO-Flag-NLS. The cDNA of the RAP80 sUIM was synthesized by GeneArt (Invitrogen) and inserted into pcDNA5-FRT-TO-Flag-NLS.

To delete the RNF168 RING domain, a fragment encompassing amino acid residues 59-571 of RNF168 was PCR-amplified and inserted into pcDNA3-GFP-NLS or pMALC2-His6. The expression vector denoted as GFP-RNF169ΔRING encompasses aa 159-708. All other deletions and point mutations in RNF168, RNF169, RNF8, the UBD-LRM2 chimeras and the RAP80 sUIM were generated using the QuikChange Mutagenesis Kit (Stratagene). Details of the MBP-His6-RNF168 and MBP-His6-RNF169 deletions are as follows: RNF168ΔMIU2, aa 439-462, RNF168ΔUBD, aa 134-194/439-462, RNF168ΔLRM1, aa 100-133, RNF169ΔMIU2, aa 662-685, RNF169ΔRING, aa 68-107. All MBP- His6-RNF168 and MBP- His6-RNF169 deletions were done in a ΔRING context. The RING deletion in pcDNA5-FRT-TO-flag-RNF169 encompasses aa 68-107.

The inducible Flp-In/T-Rex HCT116 RNF168* expression cell lines were described in (Stewart et al., 2009). All Flp-In/T-Rex HCT116 RNF169 cell lines were generated using the Flp-In T-Rex system (Invitrogen) as described in (Stewart et al., 2009). All constructs were confirmed by sequencing.

Immunofluorescence microscopy U2OS, HCT116 Flp-In T-REx or U2OS LacO SceI 111 II cells were grown on glass coverslips. Cells were fixed with 2% (w/v) paraformaldehyde in PBS for 20 min at room temperature and then permeabilized with 0.3 % (v/v) Triton X-100 for 20 min at room temperature. For RNF169 immunodetection, cells were pre-treated with nuclear extraction buffer (20 mM HEPES pH 7.5, 20 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.5% IGEPAL, Phosphatase Inhibitor Cocktail [Sigma], Protease Inhibitor Cocktail [Complete, EDTA-free; Roche]) for 20 min on ice before fixation with 2% paraformaldehyde. After fixation, cells were washed with PBS four times and then blocked with ADB (Antibody Dilution Buffer; 10 % normal goat serum, 0.1 % Triton X-100, 0.1 % saponin in PBS) for 30 min. Cells were incubated with primary antibody (diluted in ADB) for 1 h at room temperature, washed with PBS and then counterstained with Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 555 goat anti-mouse or Alexa Fluor 555 goat anti-rabbit secondary antibodies (Molecular Probes) diluted in ADB, for 1 h at room temperature. Cells were then washed twice with PBS and stained with DAPI (0.4 µg/ml in PBS) to visualize DNA. The coverslips were mounted onto glass slides with Prolong Gold mounting agent (Invitrogen). Micrograph images were taken using an inverted microscope (DMIRE2, Leica) with a spinning disk confocal scanner (CSU10, Yokogawa) and equipped with a 63X oil lens. Images were acquired using Volocity Software v. 4.1 (Improvision) and processed using Adobe Photoshop CS5. In all micrographs dashed lines indicated nucleus outlines (as determined by DAPI staining; not shown). Insets represent 3 X magnifications of the indicated fields. For analysis of 53BP1 focus morphology and quantitation of GFP fluorescence intensities, three-dimensional image data sets were acquired on an imaging system (DeltaVision Core, Applied Precision) equipped with an IX71 microscope (Olympus), a CCD camera (CoolSNAP HQ2 1024x1024; Roper Scientific), and 60x/1.42 NA objective (Olympus). For each image, 18 Z sections (0.2 μm apart) were acquired with 2 signal channels. The images were computationally deconvolved and maximum intensity projections were extracted using the SoftwoRx software package (v4.5, Applied Precision). Analysis of fluorescence intensities was performed on deconvolved, maximum-projected TIFF images using the ImageJ software (NIH) Recombinant protein production Expression of recombinant protein in E.coli strain BL21 (DE3) pLysS was induced when bacterial cultures reached an OD600 of 0.6 using 1 mM isopropyl-β-D-thiogalactopyranoside. MBP-His6 fusion proteins were first purified on Ni-NTA agarose (QIAGEN) according to the batch method described in the manufacturer’s manual (The QIAexpressionist). The eluted protein was then immobilized onto amylose resin (NEB) according to the batch method described in the manufacturer’s manual and stored in 20% glycerol. Nucleosome reconstitution Purified recombinant histones H2A (human), H2B (human), H3 (Xenopus laevis) and H4 (Xenopus laevis) were reconstituted into nucleosomes as described in (Luger et al., 1997). Isolation of chromatin from HEK293T cells To obtain chromatin-enriched fractions, HEK293T cells were lysed in EBC1 buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05% IGEPAL, 1 mM EDTA, 1 mM DTT, 1X protease inhibitors - Complete, EDTA-free, Roche) for 5 min on ice followed by centrifugation for 5 min at 1000 g. The pellet was washed once with EBC1, resuspended in EBC2 buffer (50 mM Tris-

HCl pH 7.5, 300 mM NaCl, 5mM CaCl2, 10 U micrococcal nuclease/ P150 dish (Sigma), 1X protease inhibitors - Complete, EDTA-free, Roche) and solubilized for 15 min at 30°C. The chromatin-enriched lysate was then cleared by centrifugation for 15 min at 16 000 g. MBP-RNF169/ chromatin pull-down assay 2 μg of recombinant MBP or MBP-tagged RNF169 proteins (WT, LARA and A673) were immobilized onto amylose resin (NEB) in coupling buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% NP-40, 1% BSA) overnight at 4°C. The resulting protein-amylose slurry was washed once in PD buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% NP-40, 0.1% BSA). Pull-downs were carried out by mixing the protein-amylose slurry with 400 μg of HEK293T chromatin extract (isolated from cells transiently overexpressing GFP-RNF168) for 2 h at 4°C. The amylose resin was then washed five times and eluted in 2X Laemmli Sample Buffer for analysis by immunoblotting. All incubations and washes were carried out in PD buffer. Immunoprecipitation HEK293T cells were co-transfected with the indicated GFP- and Flag-tagged expression vectors. 24 h post-transfection, cells were lysed in lysis buffer (50 mM Hepes-KOH, pH 7.5, 100 mM KCl, 2 mM EDTA, 0.5 % IGEPAL, 10% glycerol, 0.25 mM sodium orthovanadate, 10 mM NaF, 50 mM 2-glycerolphosphate, pH 7.5, 1mM DTT, 1X protease inhibitors (Complete, EDTA-free, Roche)) and cell lysates were clarified by centrifugation at 4°C. 2 mg of lysate were incubated with 15 μl of packed anti-Flag (M2) affinity gel (Sigma) for 2 h at 4°C. Beads were then washed four times with lysis buffer and eluted in 2X Laemmli Sample Buffer for analysis by immunoblotting. Isothermal Titration Calorimetry (ITC) ITC was performed using a VP-ITC calorimeter (MicroCal). MBP-tagged RNF169 proteins (aa 662-708; WT, L699A/R700R (LARA), R689A and A673G) and ubiquitin were individually dialyzed in 1X PBS and degassed. 1.8 mM ubiquitin was titrated into 60 μM MBP-RNF169 protein solution in the sample cell using 30 consecutive 10 μl injections at 25°C. Resultant binding isotherms were processed with Origin 5.0 software (Microcal). Curve fittings were carried out using the one-set-of-sites model and fixing the stoichiometry (n) to 1.

Figure S1. RNF169 Accumulates at Sites of DNA Damage Downstream of RNF168, Related to Figure 1 (A) Controls for the knockdown of BRCA1, 53BP1 and RAD18 in Figure 1C. U2OS cells were transfected with the indicated siRNAs. 48 h post-transfection, cells were irradiated and processed for immunofluorescence with the indicated antibodies. Scale bar=7 μm. (B) RNF169 is dispensable for the recruitment of RNF168, RAD18, BRCA1 and 53BP1. U2OS cells were transfected with either control (siCTRL) or RNF169 (siRNF169) siRNAs. 48 h post-transfection, cells were irradiated and processed for immunofluorescence with the indicated antibodies. Scale bar=7 μm.

(C) RNF169 IR-induced foci are dependent on the E3 ligase activity of RNF168. Flp-In/T-Rex HCT116 cell lines expressing the indicated siRNA-resistant RNF168 constructs (RNF168*) were irradiated and processed for RNF169 and γ-H2AX immunofluorescence. Scale bar=7 μm. (D) Quantitation of (C). At least 100 cells per condition were counted. Data are represented as the mean ± SD (n=3). (E) Purity of the recombinant proteins used in in vitro pull-down assays. Purified RNF168 and RNF169 constructs fused with N-terminal MBP and C-terminal hexahistidine (His6) tags were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining. MBP- His6 alone was used as control (CTRL).

Figure S2. Identification of the LRM2 in RNF168 and RNF169, Related to Figure 2 (A) Representative micrographs of the data shown in Figure 2A. U2OS cells transfected with the indicated GFP-tagged RNF168 constructs were irradiated and processed for GFP and γ-H2AX fluorescence. Scale bar = 7 μm. (B) Representative micrographs of the data shown in Figure 2B. U2OS cells were first transfected with the indicated siRNAs. 24 h later, cells were transfected with the indicated siRNA-resistant Flag-tagged RNF168 constructs (RNF168*). 24 h post-DNA transfection, cells were irradiated and processed for Flag and γ-H2AX immunofluorescence. Scale bar = 7 μm.

(C-D) Representative micrographs of the data shown in Figure 2D and Figure 2F. U2OS cells transfected with the indicated GFP-tagged RNF169 constructs were irradiated and processed for GFP and γ-H2AX fluorescence. Scale bar=7 μm. (C) Micrographs of the data shown in Figure 2D. (D) Micrographs of the data shown in Figure 2F.

Figure S3. The Observed Phenotypes Are Not Caused by Differences in Protein Expression or by Protein Oligomerization, Related to Figure 2 (A) Whole cell extracts of U2OS cells expressing the indicated GFP-tagged RNF168 constructs were separated by SDS-Page and analyzed by immunoblotting (IB). Tubulin was used as loading control. Related to Figure 2A.

(B-C) Whole cell extracts of U2OS cells expressing the indicated GFP-tagged RNF169 constructs were separated by SDS-Page and analyzed by immunoblotting (IB). Tubulin was used as loading control. (C) Related to data shown in Figure 2D. (D) Related to data shown in Figure 2F. (D) Whole cell extracts of U2OS cells expressing the indicated GFP-tagged RNF168 constructs were separated by SDS-Page and analyzed by immunoblotting (IB). Tubulin was used as loading control. Related to Figure 2H. (E) U2OS cells transfected with the indicated GFP-tagged RNF169 constructs were irradiated (10 Gy) and processed for GFP fluorescence. The integrated nuclear fluorescence intensity of at least 40 GFP-positive cells was quantified using ImageJ software (NIH). (F-G) HEK293T whole cell extracts expressing the indicated Flag- and GFP-tagged RNF168 and RNF169 constructs, respectively, were subjected to immunoprecipitation with anti-Flag affinity gel and analyzed by immunoblotting (IB) with the indicated antibodies. (H) Isothermal titration calorimetry profiles obtained by titration of ubiquitin against recombinant MBP-tagged RNF169 (aa 662-708; WT, L699A/R700A (LARA), R689A or A673G) proteins. The dissociation constants (Kd) obtained by fitting to a one-site binding model are shown in the insets. The purity of the MBP-fusion proteins is shown in Figure S5E.

Figure S4. Identification of the LRM1 in RNF168, Related to Figure 4 Representative micrographs of the data shown in Figure 4A. U2OS cells were first transfected with the indicated siRNAs. 24 h later, cells were transfected with the indicated siRNA-resistant GFP-tagged RNF168 constructs. 24 h post-DNA transfection, cells were irradiated and processed for GFP and γ-H2AX immunofluorescence. Scale bar=7 μm.

Figure S5. Targeting mCherry-LacR-RNF168 to Chromatin Results in 53BP1 Recruitment, Related to Figure 5

(A-B) U2OS LacO SceI 111 II cells were transfected with the indicated mCherry-LacR-RNF168 (LacR-RNF168) constructs and processed for mCherry and 53BP1 or FK2 immunofluorescence. Representative micrographs are shown in the left panel and quantitation is shown in the right panel. At least 100 arrays were counted. Data are represented as the mean ± SD (n=5), scale bar=5 μm. (C) Alignment of RNF168 homologs. RNF168 sequences from the indicated species were downloaded from UNIPROT and aligned using the Clustal Omega multiple sequence alignment (http://www.ebi.ac.uk/Tools/msa/clustalo/) with default parameters. The output alignment was then pasted into Boxshade 3.21 (http://www.ch.embnet.org/) and the output .rtf file was edited to keep only the region corresponding to residues 110-170 of human RNF168. (D) Slot-blot of the RNF169 LRM2 peptides used in Figures 5D and 5F. 100 ng of each biotinylated peptide (scrambled control or RNF169 WT, L699A/R700A (LARA) and R689A) were immobilized by slot-blotting and were detected using ExtrAvidin (EA) coupled to HRP. The numbers indicate relative band intensities as determined by densitometry using ImageJ software (NIH). (E) Purity of the recombinant proteins used in Figures 5D and S3H. Purified RNF169 proteins (aa 662-708; WT, L699A/R700A (LARA), A673G or R689A) fused with N-terminal MBP and C-terminal hexahistidine (His6) tags were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue (CBB) staining. MBP-His6 alone was used as control (CTRL).

Figure S6. siRNA-Mediated Depletion of RNF169 alters 53BP1 Focus Morphology, Related to Figure 6

(A) U2OS cells were transfected with the indicated siRNAs. 48 h post-transfection, cells were either irradiated and processed for 53BP1 immunofluorescence (left panel; at least 100 cells per condition were counted. Data are represented as the mean ± SD (n=3)) or lysed and analyzed by immunoblotting with RNF169 and tubulin antibodies (right panel). (B) U2OS cells were transfected with the indicated siRNAs. 48 h post-transfection, cells were processed for 53BP1 immunofluorescence. DNA was counterstained with DAPI.

Figure S7. Identification of LRM Motifs in RAD18 and RAP80, Related to Figure 7

(A) Representative micrographs of the data shown in Figure 7B. U2OS cells transfected with the indicated GFP-tagged RAD18 constructs were irradiated and processed for GFP and γ-H2AX fluorescence. Scale bar=7 μm. (B) HEK293T whole cell extracts expressing the indicated Flag- and GFP-tagged RAD18 constructs were subjected to immunoprecipitation with anti-Flag affinity gel and analyzed by immunoblotting (IB) with the indicated antibodies. (C) Representative micrographs of the data shown in Figure 7D. U2OS cells transfected with the indicated GFP-tagged RAP80 constructs were irradiated and processed for GFP and γ-H2AX fluorescence. Scale bar=7 μm.

SUPPLEMENTAL REFERENCES Kolas, N.K., Chapman, J.R., Nakada, S., Ylanko, J., Chahwan, R., Sweeney, F.D., Panier, S., Mendez, M., Wildenhain, J., Thomson, T.M., et al. (2007). Orchestration of the DNA-Damage Response by the RNF8 Ubiquitin Ligase. Science 318, 1637-1640. Luger, K., Rechsteiner, T.J., Flaus, A.J., Waye, M.M., and Richmond, T.J. (1997). Characterization of nucleosome core particles containing histone proteins made in bacteria. J Mol Biol 272, 301-311. Stewart, G.S., Panier, S., Townsend, K., Al-Hakim, A.K., Kolas, N.K., Miller, E.S., Nakada, S., Ylanko, J., Olivarius, S., Mendez, M., et al. (2009). The RIDDLE Syndrome Protein Mediates a Ubiquitin-Dependent Signaling Cascade at Sites of DNA Damage. Cell 136, 420-434.