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S41467-018-04466-4.Pdf ARTICLE DOI: 10.1038/s41467-018-04466-4 OPEN Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets Dragomir B. Krastev1, Stephen J. Pettitt 1, James Campbell1, Feifei Song1, Barbara E. Tanos2, Stoyno S. Stoynov3, Alan Ashworth1,4 & Christopher J. Lord1 Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events 1234567890():,; that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor “tagged” cells in a genetic screen we carry out a large-scale identifi- cation of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites. 1 The CRUK Gene Function Laboratory and Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London SW3 6JB, UK. 2 The Cancer Therapeutics Division, The Institute of Cancer Research, London SW3 6JB, UK. 3 The Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. 4Present address: UCSF Helen Diller Family Comprehensive Cancer Center, 1450 3rd Street, San Francisco, CA 94158, USA. Correspondence and requests for materials should be addressed to A.A. (email: [email protected]) or to C.J.L. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:2016 | DOI: 10.1038/s41467-018-04466-4 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04466-4 oly ADP-ribosylation (PARylation) is a highly dynamic and PBZ refers to the APLF domain and CHFR-PBZ will be explicitly Preversible post-translation protein modification that is written when it is used). We then compared the ability of PAR generated by a family of PAR polymerases (PARPs, biosensors to detect DNA damage-induced PAR, when compared ARTDs). The PARP superfamily encompasses 17 proteins, of to PAR immunodetection with a commonly used anti-PAR which only PARP1, 2, and tankyrases (TNKS and TNKS2, also antibody (10H). To elicit DNA damage-induced PARylation, we 1 known as PARP5a and 5b) display a clear PARP activity . The exposed HeLa cells to H2O2; to reduce PAR, we exposed cells to remaining family members are mono ADP-ribose transferases or the PARP1/2 inhibitor olaparib. In untreated cells, cells exposed lack enzymatic activity. PARP1, 2, and 3 are nuclear proteins to H2O2, or cells exposed to olaparib (Fig. 1b), the antibody and involved in DNA damage responses (DDR)2, while tankyrases the biosensor signals were correlated (Spearman’s rank correla- = regulate a variety of cellular processes including telomere main- tion, rs 0.48, 0.53, and 0.39 respectively) suggesting that the tenance3, Wnt signaling4 and mitotic progression5. The role of biosensor signal recapitulated the detection of PARylation shown PARP1, 2, and 3 in the DDR provided the rationale for the dis- by immunodetection (exemplary images are shown in Supple- covery and development of clinical PARP inhibitors. In addition, mentary Fig. 1a). H2O2 exposure caused a close to twofold tankyrase inhibition can suppresses constitutive Wnt signaling4, increase in 10H PAR signal compared to the basal state, while which has led to the discovery of a series of small molecule olaparib treatment did not lead to any significant decrease in PAR TNKS/TNKS2 inhibitors78. Given the burgeoning interest in the signal (Fig. 1c), consistent with the notion that the 10H antibody PARP superfamily enzymes as drug targets and their role as predominately recognizes long damage-induced PAR chains, but mediators of cellular signaling processes, identifying and char- fails to detect endogenous PARylation. In contrast, the PAR acterizing the targets of these enzymes is critical. biosensor revealed a broader dynamic range; H2O2 exposure A number of studies have identified PARylation targets en caused an above fivefold increase in the nuclear GFP intensity masse by isolating proteins that bind to either anti-PAR anti- (Fig. 1c), while olaparib treatment led to a twofold decrease, bodies or PAR-binding protein domains and identifying these by resulting in a 12-fold difference between the absence and damage- mass-spectrometry9. PARylation is often a transient modification, induced PAR levels. therefore, some studies have used exposure to DNA-damaging We also assessed the ability of a PAR biosensor to monitor agents to enhance DNA damage-dependent PARylation, or sup- temporal changes in PARylation. To do this, we used PBZ- pression of PAR glycohydrase (PARG) to prevent PAR degra- mRuby2 biosensor alongside a PARP1-GFP expression construct; dation10. An additional complication of such studies is that this allowed us to temporally co-monitor PARP1 and PAR PARylation is often induced on non-specific targets during localization on ultraviolet microirradiated regions of cells. To in vitro cell lysis9, 11. Hence, additional approaches to detect and eliminate any potential interference from endogenous PARP1, we – – characterize PARylation targets are required. carried out these experiments in PARP1 / cells (Methods; In this study, we describe a system for identifying and char- Fig. 1d). Localized laser microirradiation led to a rapid acterizing PARylation events that exploits the ability of PBZ localization of PARP1 to the site of DNA damage (within 200 (PAR-binding zinc finger) domains to bind PAR with high- ms), followed 300 ms later by localization of PAR at the same site affinity. By linking PBZ domains to bimolecular fluorescent (Fig. 1e). After 30–60 s, both the PARP1-GFP and PBZ-mRuby2 complementation (BiFC) biosensors, we developed fluorescent signals were reduced in a co-ordinated fashion (Fig. 1f), likely PAR biosensors that allow the detection and localization of reflecting reduction of PARP1 localization and activity after the PARylation events in live cells. Finally, by exploiting transposon- initial stages of DNA repair. The binding of the biosensor to the mediated recombination, we integrated these PAR biosensors en microirradiated site was rapid and reversible as shown by FRAP masse into thousands of protein coding genes in living cells. experiments (Supplementary Fig. 1c). To confirm the specificity Using these PAR-biosensor “tagged” cells in a genetic screen of this effect, we used two mutant PARP1-GFP fusions Fig. 1g): a facilitates the large-scale identification of PARylation targets. DNA-binding deficient mutant of PARP1 with mutations of Using this approach, we show that CTIF (CBP80/CBP20- residues 43 and 44 that disrupt the ZnF1 domain (PARP1-p. dependent translation initiation factor) is a target of PARylation [43delM;44 F > I]13); or PARP1 with an E988K mutation that by tankyrases at centrosomes and plays a role in the distribution impairs catalytic activity. The PBZ-mRuby2 sensor signal at of the centrosomal satellites. microirradiated regions was entirely dependent upon wild-type PARP1 (Fig. 1h). Exposure of HeLa cells to the clinical PARP inhibitor talazoparib also abolished the PBZ-mRuby2 sensor Results signal (Fig. 1i). Importantly, in this experiment PARP1-GFP was PAR-binding domains as high-affinity cellular biosensors.We expressed at endogenous levels from a bacterial artificial aimed to develop a set of PAR-biosensors that could detect chromosome (BAC) showing that PARP1 overexpression does PARylation events in living cells. To do this, we exploited the not alter the behavior of the biosensor. Furthermore, using HeLa PAR-binding ability of PBZ (PAR-binding zinc finger) domains cells without any additional PARP1 expression, we assessed the derived from either APLF (aprataxin PNK-like factor) or CHFR behavior of PBZ-mRuby2 and CHFR-PBZ-GFP (Supplementary (checkpoint protein with FHA and RING domains) to bind PAR Fig. 1b). Both biosensors showed identical kinetics, confirming with high-affinity12. Although several other PAR-binding that the observed results are due to the dynamics of PAR domains exist (such as macro and WWE domains), we selected modification rather than the specificities of the PBZ domain used. PBZ domains for the development of biosensors for the following Taken together, this data suggested that the PAR biosensors we reasons: (i) their well-defined structure with the possibility to developed exhibited high sensitivity and PAR-dependent beha- engineer precise point mutations that abolish PAR binding12, and vior and could be used to dynamically monitor the amount of (ii) their intermediate PAR-binding affinity (weaker compared to cellular PARylation. macrodomains), which allows reversible binding (this is con- firmed below), thus minimizing the possibility of artefactual PAR stabilization and interference with endogenous PARylation- Development and validation of BiFC biosensors of PARylation. dependent processes. We fused the coding sequence of the The identification of PARylation targets via biochemical pur- APLF or CHFR-PBZ domain to that of green fluorescent protein ification is confounded by the artefactual loss and gain of PAR- (GFP), generating
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