SHLD 2/ FAM 35A co‐operates with REV 7 to coordinate DNA double‐strand break repair pathway choice

Article28 August 2018Open Access Transparent process SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice Steven Findlay Steven Findlay Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author John Heath John Heath Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Vincent M Luo Vincent M Luo Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada Search for more papers by this author Abba Malina Abba Malina orcid.org/0000-0002-8511-9005 Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Théo Morin Théo Morin Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Yan Coulombe Yan Coulombe Genome Stability Laboratory, CHU de Québec Research Center, Quebec City, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec City, QC, Canada Search for more papers by this author Billel Djerir Billel Djerir Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Zhigang Li Zhigang Li Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Arash Samiei Arash Samiei Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Estelle Simo-Cheyou Estelle Simo-Cheyou Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Martin Karam Martin Karam Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Halil Bagci Halil Bagci Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada Search for more papers by this author Dolev Rahat Dolev Rahat Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Damien Grapton Damien Grapton Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Elise G Lavoie Elise G Lavoie Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Christian Dove Christian Dove Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Husam Khaled Husam Khaled Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Hellen Kuasne Hellen Kuasne Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Koren K Mann Koren K Mann Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Kathleen Oros Klein Kathleen Oros Klein Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Celia M Greenwood Celia M Greenwood Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Department of Epidemiology, Biostatistics and Occupational Health, MGill University, Montreal, QC, Canada Search for more papers by this author Yuval Tabach Yuval Tabach Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Morag Park Morag Park Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Jean-Francois Côté Jean-Francois Côté orcid.org/0000-0001-7055-2642 Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, QC, Canada Département de Médecine (Programmes de Biologie Moléculaire), Université de Montréal, Montreal, QC, Canada Search for more papers by this author Jean-Yves Masson Jean-Yves Masson Genome Stability Laboratory, CHU de Québec Research Center, Quebec City, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec City, QC, Canada Search for more papers by this author Alexandre Maréchal Alexandre Maréchal Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Alexandre Orthwein Corresponding Author Alexandre Orthwein [email protected] orcid.org/0000-0002-7350-3413 Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Steven Findlay Steven Findlay Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author John Heath John Heath Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Vincent M Luo Vincent M Luo Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada Search for more papers by this author Abba Malina Abba Malina orcid.org/0000-0002-8511-9005 Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Théo Morin Théo Morin Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Yan Coulombe Yan Coulombe Genome Stability Laboratory, CHU de Québec Research Center, Quebec City, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec City, QC, Canada Search for more papers by this author Billel Djerir Billel Djerir Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Zhigang Li Zhigang Li Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Arash Samiei Arash Samiei Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Estelle Simo-Cheyou Estelle Simo-Cheyou Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Martin Karam Martin Karam Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Halil Bagci Halil Bagci Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada Search for more papers by this author Dolev Rahat Dolev Rahat Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Damien Grapton Damien Grapton Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Elise G Lavoie Elise G Lavoie Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Christian Dove Christian Dove Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Husam Khaled Husam Khaled Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Search for more papers by this author Hellen Kuasne Hellen Kuasne Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Koren K Mann Koren K Mann Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Kathleen Oros Klein Kathleen Oros Klein Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Search for more papers by this author Celia M Greenwood Celia M Greenwood Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Department of Epidemiology, Biostatistics and Occupational Health, MGill University, Montreal, QC, Canada Search for more papers by this author Yuval Tabach Yuval Tabach Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israel Search for more papers by this author Morag Park Morag Park Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada Search for more papers by this author Jean-Francois Côté Jean-Francois Côté orcid.org/0000-0001-7055-2642 Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, QC, Canada Département de Médecine (Programmes de Biologie Moléculaire), Université de Montréal, Montreal, QC, Canada Search for more papers by this author Jean-Yves Masson Jean-Yves Masson Genome Stability Laboratory, CHU de Québec Research Center, Quebec City, QC, Canada Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec City, QC, Canada Search for more papers by this author Alexandre Maréchal Alexandre Maréchal Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada Search for more papers by this author Alexandre Orthwein Corresponding Author Alexandre Orthwein [email protected] orcid.org/0000-0002-7350-3413 Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada Division of Experimental Medicine, McGill University, Montreal, QC, Canada Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada Search for more papers by this author Author Information Steven Findlay1,2,‡, John Heath1,2,‡, Vincent M Luo1,3, Abba Malina1,4, Théo Morin5, Yan Coulombe6,7, Billel Djerir5, Zhigang Li1, Arash Samiei1,2, Estelle Simo-Cheyou1,4, Martin Karam2, Halil Bagci8,9, Dolev Rahat10, Damien Grapton1, Elise G Lavoie1, Christian Dove1,2, Husam Khaled1,2, Hellen Kuasne11, Koren K Mann1,2,4, Kathleen Oros Klein1, Celia M Greenwood1,12, Yuval Tabach10, Morag Park11, Jean-Francois Côté8,9,13,14, Jean-Yves Masson6,7, Alexandre Maréchal5 and Alexandre Orthwein *,1,2,3,4 1Lady Davis Institute for Medical Research, Segal Cancer Centre, Jewish General Hospital, Montreal, QC, Canada 2Division of Experimental Medicine, McGill University, Montreal, QC, Canada 3Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada 4Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada 5Department of Biology, Université de Sherbrooke, Sherbrooke, QC, Canada 6Genome Stability Laboratory, CHU de Québec Research Center, Quebec City, QC, Canada 7Department of Molecular Biology, Medical Biochemistry and Pathology, Laval University Cancer Research Center, Quebec City, QC, Canada 8Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada 9Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada 10Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, The Hebrew University of Jerusalem, Jerusalem, Israel 11Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada 12Department of Epidemiology, Biostatistics and Occupational Health, MGill University, Montreal, QC, Canada 13Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, QC, Canada 14Département de Médecine (Programmes de Biologie Moléculaire), Université de Montréal, Montreal, QC, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 514 340 8222; E-mail: [email protected] The EMBO Journal (2018)37:e100158https://doi.org/10.15252/embj.2018100158 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract DNA double-strand breaks (DSBs) can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)-based approach, we identify 11 high-confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ-mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1-, RIF1-, and REV7-dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision-making process during DSB repair. Synopsis Proteomic interaction and CRISPR/Cas9 doxorubicin sensitivity screens identify shieldin proteins as the sought-after effectors through which the 53BP1/RIF1/REV7 axis antagonizes homologous recombination (HR) and promotes non-homologous end-joining (NHEJ) repair of DNA double strand breaks (DSB). Mass spectrometry identifies SHLD2/FAM35A as REV7 interactor. SHLD2 is recruited to DSBs and promotes their repair. SHLD2 promotes NHEJ and antagonizes HR by inhibiting DNA end resection. SHLD2 is part of a larger multi-protein complex composed of REV7 and SHLD1/C20orf196. SHLD2 is a biomarker for the prognosis of TNBC/basal-like BC. Introduction Due to their highly recombinogenic and pro-apoptotic potential, DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions. Their inaccurate resolution can result in point mutations, small deletions/insertions, chromosomal rearrangements, or loss of gross genetic information that drive genomic instability, carcinogenesis, and cell death (reviewed in Tubbs & Nussenzweig, 2017). To avoid these deleterious outcomes, cells have deployed a complex network of proteins to signal and repair DSBs. One critical step during the DSB response consists in the choice between two mutually exclusive DNA repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR; reviewed in Ceccaldi et al, 2016). This decision process, named DNA repair pathway choice, integrates several elements including the cell cycle status, the complexity of the DNA end, and the epigenetic context. Importantly, DNA repair pathway choice is under the control of two antagonizing factors, 53BP1 and BRCA1 (reviewed in Hustedt & Durocher, 2016). Non-homologous end-joining is predominantly involved in the repair of DSBs during the G1 phase of the cell cycle. It is characterized by a limited processing of the DNA ends catalyzed by the nuclease Artemis and their subsequent ligation by DNA ligase IV (reviewed in Betermier et al, 2014). Importantly, NHEJ is promoted by the recruitment of 53BP1 at DSBs, along with its effectors RIF1, REV7, and PTIP (Chapman et al, 2012, 2013; Callen et al, 2013; Di Virgilio et al, 2013; Escribano-Diaz et al, 2013; Feng et al, 2013; Zimmermann et al, 2013; Boersma et al, 2015; Xu et al, 2015). These latter factors play a central role in several additional biological processes, including the establishment of a protective immunity during class switch recombination (CSR), a programmed DSB-dependent process that specifically occurs in B cells (Manis et al, 2004; Ward et al, 2004; Chapman et al, 2013; Di Virgilio et al, 2013; Escribano-Diaz et al, 2013; Boersma et al, 2015; Xu et al, 2015). In S/G2 phases of the cell cycle (when sister chromatids are available as templates), HR is activated and can alternatively repair DSBs. One of the key features of HR is the formation of long stretches of single-stranded DNA (ssDNA), a process called DNA end resection (reviewed in Fradet-Turcotte et al, 2016). The resulting ssDNA stretches are rapidly coated by RPA, which is subsequently replaced by the recombinase RAD51 to form nucleofilaments that are a pre-requisite for the subsequent search of homology, strand invasion, and strand exchange before the resolution of the DSB by the HR machinery. Critically, BRCA1 promotes the initiation of DNA end resection and HR-mediated DSB repair by preventing the recruitment of 53BP1 and its downstream effectors to sites of DNA damage in S/G2 phases (Chapman et al, 2012, 2013; Escribano-Diaz et al, 2013; Feng et al, 2013), thereby antagonizing 53BP1 function in NHEJ. While the opposing role of 53BP1 and BRCA1 in DNA repair pathway choice has been extensively scrutinized over the past years, it remains largely unclear how the 53BP1 downstream effectors, namely REV7, promote NHEJ and antagonize BRCA1-mediated HR in G1 phase of the cell cycle (Boersma et al, 2015; Xu et al, 2015). REV7 is an adaptor protein that has been described for its role in mitotic progression through the control of both the activity of the spindle assembly checkpoint (SAC) and the formation of a functional anaphase-promoting complex/cyclosome-Cdc20 (APC/C; Cahill et al, 1999; Listovsky & Sale, 2013; Bhat et al, 2015). In parallel, REV7 is a well-defined player in DNA translesion synthesis (TLS; reviewed in Waters et al, 2009) as well as DSB repair by HR as part of a complex composed of the deoxycytidyl (dCMP) transferase REV1 and the catalytic subunit of the DNA polymerase ζ, REV3L (Sharma et al, 2012). The recent discovery that REV7 participates in the NHEJ pathway in a TLS-independent manner raised fundamental questions about how this adaptor protein promotes DSB repair and controls DNA repair pathway choice. In this present study, we sought to get insight into the decision-making process underpinning DNA repair pathway choice by deciphering the interactome of REV7. Using a mass spectrometry (MS)-based approach, we identified SHLD2 (previously known as FAM35A/RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A accumulates at DSBs through its N-terminal domain in a 53BP1-, RIF1-, and REV7-dependent manner. Importantly, depletion of FAM35A impairs both NHEJ and CSR, while promoting DNA end resection and HR. In fact, FAM35A acts in concert with SHLD1 (previously known as C20orf196/RINN3) in promoting both NHEJ and CSR while antagonizing HR. Altogether, our results provide a better insight into the molecular events that control DNA repair pathway choice. Results Mapping of REV7 proximal/interacting partners relevant for DNA repair pathway choice To get better insight into the interactome of REV7, we performed a standard affinity purification (AP) followed by MS (AP-MS; Fig EV1A), where REV7 was tagged with the Flag epitope and stably expressed in the human embryonic kidney 293 (HEK293) cell line using the Flp-In/T-REX system (Fig EV1B). As a complementary approach, we used a proximity-based biotin labeling technique (BioID), which allows the monitoring of proximal/transient interactions (Fig EV1A; Roux et al, 2012, 2013). Briefly, REV7 was fused to a mutant of an Escherichia coli biotin-conjugating enzyme (BirA*) and stably expressed in HEK293 as previously described (Lambert et al, 2015). This fusion protein is capable of biotinylating proteins that come in close proximity or directly interact with REV7 (Fig EV1C). Labeled proteins were subsequently purified by streptavidin affinity and identified by MS. Both approaches were carried out in triplicate using extracts of cells treated in the absence or the presence of the radiomimetic DNA damaging drug neocarzinostatin (NCS). We identified 140 high-confidence REV7 interactors that were either common to the four experimental conditions or found in both the AP-MS and the BioID following NCS treatment (Fig 1A and Table EV1). As expected, pathways critical for mitosis and DNA repair were enriched in our list of REV7 partners (Fig EV1D). To further refine REV7 interactors, we intersected our data with previously reported proteomic profiling of REV7 (Nelson et al, 1999; Chen & Fang, 2001; Weterman et al, 2001; Guo et al, 2003; Iwai et al, 2007; Zhang et al, 2007; Hong et al, 2009; Medendorp et al, 2009; Tissier et al, 2010; Vermeulen et al, 2010; Listovsky & Sale, 2013; Rolland et al, 2014; Huttlin et al, 2015, 2017). Using this methodology, we obtained 11 high-confidence REV7 interactors (Figs 1B and EV1E), including the chromosome alignment-maintaining phosphoprotein (CHAMP1), a kinetochore-microtubule attachment protein that has been recently linked to REV7 and its role during mitotic progression (Itoh et al, 2011), and the cell-division cycle protein 20 (CDC20), a critical activator of the anaphase-promoting complex (APC/C) that allows chromatid separation and entrance into anaphase (Chen & Fang, 2001; Listovsky & Sale, 2013; Bhat et al, 2015). However, whether these high-confidence REV7 interactors play any role in the DSB response remains unresolved. Click here to expand this figure. Figure EV1. Analysis of REV7 interactome Schematic representation of AP-MS stable interaction Flag-REV7 pull-down and the BioID BirA*-REV7 biotinylation of proximal interactors. HEK293-TREx cells stably expressing an inducible Flag-REV7 construct were tested for expression following induction with tetracycline as indicated. After lysis, samples were immunoblotted for FLAG and REV7. Actin was used as a loading control. HEK293-TREx cells stably expressing an inducible BirA-Flag or BirA-Flag-REV7 construct were tested for expression and biotinylation following induction with tetracycline and incubation with biotin as indicated. After lysis, samples were immunoblotted for FLAG and streptavidin. Actin was used as a loading control. The interactome of REV7 obtained from both the AP-MS and the BioID approaches was analyzed for pathway enrichment using EnrichR. The y-axis represents the ratio of the number of genes from the dataset that map to the pathway and the number of all known genes ascribed to the pathway and is defined as enrichment of P-value (−log10). Network representation of the selected 11 high-confidence interactors of REV7 (annotated as MAD2L2 in this figure) and their previously described interactors. Proteins are represented following the k-means clustering through STRING v10.5. Cell cycle distribution of U2OS EJ5-GFP cells transfected with the indicated siRNAs and subsequently for propidium iodide (PI) staining and flow cytometry analysis. Data are presented as the mean (n = 2). Download figure Download PowerPoint Figure 1. Identification of novel REV7 interactors relevant for the NHEJ pathway Venn Diagram representing the distribution of proteins identified by both the BioID and the standard AP/MS of REV7, with or without DNA damage (NCS). Selected BioID REV7 results, shown as dot plots. The spectral counts for each indicated prey protein are shown as AvgSpec. Proteins were selected based on and iProphet probability of > 0.95, BFDR of < 0.05, and ≥ 10 peptide count. The circle size represents the relative abundance of preys over baits. U2OS EJ5-GFP cells were transfected with the indicated siRNAs. At 24 h post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48-h post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL). Data are presented as the mean ± SD (n = 3). Significance was determined by one-way ANOVA followed by a Dunnett's test. *P < 0.05, **P < 0.005, ***P < 0.0005. Download figure Download PowerPoint To ascertain the relevance of these interactors for NHEJ, we used a well-established GFP-based reporter assay that monitors total NHEJ events (Bennardo et al, 2008), the EJ5-GFP assay, and targeted each candidate using small interfering RNA (siRNA) pools (Fig 1C). As positive controls, we incorporated both RIF1 and REV7, which have been previously shown to impair this assay (Chapman et al, 2013; Escribano-Diaz et al, 2013; Boersma et al, 2015). Out of the 11 candidates tested, downregulation of seven REV7 interactors significantly impaired the restoration of the GFP signal following DSB induction and subsequent repair by NHEJ (Fig 1C), without impacting drastically cell cycle progression (Fig EV1F). SHLD2 emerged as our strongest hit, with a reduction of more than 60% of the GFP signal compared to the control condition in this assay (Fig 1C). Therefore, we concentrated our efforts on this factor to better define its involvement in DNA repair pathway choice. SHLD2 promotes DSB repair in human cells To get an evolutionary perspective and determine whether SHLD2 may be relevant for DNA repair, we used a novel phylogenetic profiling (PP) approach and defined the landscape of genes that co-evolved with SHLD2 among mammals and vertebrates (Tabach et al, 2013a). Importantly, this PP method has been previously shown to successfully predict protein function by analyzing the genes that co-evolved with a given factor of interest (Tabach et al, 2013a,b). Gene ontology analysis for biological process enrichment identified DNA repair, IL-1 signaling, Nucleotide Excision repair (NER), and the APC/C-CDC20 pathway as the most significant biological functions associated with genes that co-evolved with SHLD2 (Fig 2A). Strikingly, SHLD2 co-evolves with RIF1 in both mammalians and vertebrates, which further suggests a putative role of SHLD2 in the maintenance of genome stability. Figure 2. SHLD2 plays a critical role in DNA repair Pathway enrichment analysis on genes co-evolving with SHLD2 in Mammalians and Vertebrates using a phylogenetic profiling approach followed by an Enrichr-based analysis. Quantification of the Neutral Comet assay. U2OS cells stably expressing shCtrl, shREV7, or shSHLD2 were exposed to IR (10 Gy) and run in low melting agarose under neutral conditions. Immunofluorescence against DNA stained with SYBR Gold was performed to measure the tail moment. Data are represented as a box and whiskers graph where the box tends from the 25th to the 75th percentiles, while the whiskers are