Volume 52, Issue 2, Pages 272-285 (October 2013) Proteome-wide Identification of Poly(ADP-Ribosyl)ation Targets in Different Genotoxic Stress Responses  Stephanie Jungmichel, Florian Rosenthal, Matthias Altmeyer, Jiri Lukas, Michael O. Hottiger, Michael L. Nielsen  Molecular Cell  Volume 52, Issue 2, Pages 272-285 (October 2013) DOI: 10.1016/j.molcel.2013.08.026 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Proteome-wide Identification of PARylated Proteins in Response to Genotoxic Stress with SILAC-Based Quantification (A) A schematic representation of the SILAC-based enrichment strategy. U2OS cells were grown in light, medium, or heavy SILAC medium. Heavy-labeled cells were treated with genotoxic stress. Separate pull-downs of SILAC-encoded lysates were performed with a PAR-binding-defective Af1521 mutant (G42E) for light SILAC state and a PAR-binding GST-Af1521 wild-type macrodomain for medium and heavy SILAC states. Eluates were resolved by SDS-PAGE and analyzed by high-resolution LC-MS/MS. (B) Quantitative comparison of tryptic peptide abundances. (C) SILAC-based enrichment of PARylated proteins in the absence of exogenous genotoxic stress. Logarithmized H/M SILAC ratios from the forward and M/H SILAC ratios from the reverse experiment were plotted against each other. Regulated proteins reveal strong enrichment of proteins involved in DNA repair processes in comparison to annotated GO genes across the entire human genome (indicated p values < 0.005). (D) U2OS cell lysate preparation in the absence or presence of 3-AB in the lysis buffer to prevent unphysiological activation of protein PARylation and cells treated with 3-AB prior to cell lysis (lane 4). Pull-downs were performed with GST-Af1521_wt and analyzed by WB with the indicated antibodies. (E) Hierarchical clustering of proteins derived from SILAC-based U2OS lysate pull-down in the absence (−) or presence (+) of 3-AB (5 mM) or PJ-34 in the lysis buffer. Functional GO analysis of regulated proteins in the absence of PARPi reveals strong enrichment of PARylated proteins involved in DNA repair. (F) U2OS cells were transfected with PARG siRNA for 72 hr and treated with H2O2 and analyzed by WB with PAR antibody. Molecular Cell 2013 52, 272-285DOI: (10.1016/j.molcel.2013.08.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Quantitative Analysis of DNA-Damage-Induced Protein PARylation (A) Four biological SILAC experiments were performed in order to investigate the impact of different DNA-damaging agents on protein PARylation. In the single experiments, cells were treated in heavy SILAC state with H2O2 (1 mM, 10 min), MMS (10 mM, 1 hr), UV (40 J/m2, 1 hr), or IR (10 Gy, 1 hr) respectively. All pull-downs were performed with siPARG-treated U2OS cells and 40 μM PJ-34 and 1 μM PARG inhibitor ADP-HPD in the lysis buffer. (B) Significantly upregulated PARylated proteins in response to oxidative stress. Logarithmized M/L and H/L SILAC ratios from the H2O2 experiment were plotted, representing untreated and treated cells. M/L SILAC ratios cumulate around 1 and follow a normal distribution, whereas H/L ratios are shifted upwardly, corresponding to induced protein PARylation. Statistical significance was calculated with a Wilcoxon rank-sum test. (C) Box plot analysis of logarithmized M/L SILAC ratios from all four SILAC experiments (see A). No regulation of PARylated proteins is observed in the absence of genotoxic stress (whiskers with minimum and maximum 1.5 interquartile range [IQR]). (D) Box plot analysis of logarithmized H/L SILAC ratios. Significant regulation reveals a strong induction of protein PARylation upon genotoxic stress (whiskers with minimum and maximum 1.5 IQR). (E) SiPARG-transfected cells were treated with DNA-damaging agents and analyzed by WB with the indicated antibodies. The extent of protein PARylation follows the distribution of SILAC ratios in (D). (F) A heat map of quantified proteins from the combined pull-downs in the absence (−) or presence (+) of DNA damage. PARylated proteins induced by genotoxic stress are clustered together. (G) GO term annotation enrichment for cellular distribution of significantly upregulated proteins in all four SILAC experiments. Molecular Cell 2013 52, 272-285DOI: (10.1016/j.molcel.2013.08.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Identification and In Vitro Validation of Protein PARylation Targets (A) Overlap of total number of PARylated proteins identified in this study with proteins that were previously observed to be PARylated and assigned with a role in DNA repair on the basis of GO term annotation. (B) In vitro PARylation of identified protein targets in the SILAC screen. Purified full-length human ARTD1/PARP1 (left) and ARTD2/PARP2 (right) were incubated with recombinantly expressed proteins in the presence of [32P]-NAD and double-stranded DNA oligomer. Samples were resolved by SDS-PAGE and analyzed by Coomassie blue (bottom) and autoradiography (AR, top). ∗, ARTD1/PARP1; ∗∗, ARTD2/PARP2; Ctrl, control including Histone H1 and zinc finger 1 and 2 from ARTD1/PARP1. (C) ARTD1/PARP1 accounts for the majority of protein PARylation in response to genotoxic stress. U2OS cells were cotransfected with PARG siRNA and either control, ARTD1/PARP1, or ARTD2/PARP2 siRNA. Cells were treated with DNA-damaging agents as described in Figure 2A and analyzed by WB with the indicated antibodies. Molecular Cell 2013 52, 272-285DOI: (10.1016/j.molcel.2013.08.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 In Vivo PARylation of Protein Targets (A) siPARG-transfected U2OS cells were incubated with PJ-34 (40 μM, 1.5 hr) or left untreated prior to treatment with H2O2 (1 mM, 10 min) as indicated. Cell lysates were pulled down with GST-Af1521_wt and analyzed by WB with the indicated antibodies in order to detect enriched PARylated proteins. NF-κB antibody was used as negative control. (B) HeLa cells stably expressing TAF15, THRAP3, or SMARCA5 as GFP fusion proteins were transfected with PARG siRNA and treated with PJ-34 and H2O2 as in (A). Lysates were subjected to Af1521_wt pull-down or GFP immunoprecipitation and subsequently analyzed by WB with PAR and GFP antibodies (∗, PARylated GFP target protein). Molecular Cell 2013 52, 272-285DOI: (10.1016/j.molcel.2013.08.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Functional Consequences of Distinct Types of DNA Damage Treatment on Protein PARylation (A) Protein interaction networks of significantly PARylated proteins from all SILAC experiments grouped into DNA metabolism by GO term annotation for biological process (p < 8 × 10−14). Network interaction data were extracted from the STRING database and visualized with Cytoscape. (B) InterPro domain annotation associated with the molecular function of significantly regulated proteins from all SILAC experiments in comparison to annotated GO genes in the entire genome (indicated p values < 1.5× 10−17 ). Strong enrichments for RNA- and DNA-binding proteins are observed. (C) A Venn diagram demonstrating the overlap of PARylated proteins from the individual DNA damage experiments (as described in Figure 2A). (D) GO enrichment analysis reveals specific enrichment of RNA metabolic processes in H2O2 and MMS experiment, whereas proteins involved in DNA metabolic processes are equally identified across all DNA damage experiments. Analysis was performed with GO term annotations for biological processes of significantly regulated protein sets in each DNA damage SILAC experiment (specific p values are listed in Figure S4C). (E) Protein interaction networks of significantly PARylated proteins from the H2O2 (top) and MMS (bottom) experiments grouped into RNA metabolism by GO term annotation for biological process (p < 2 ×10−16). Molecular Cell 2013 52, 272-285DOI: (10.1016/j.molcel.2013.08.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 PARylation-Dependent Relocalization of TAF15 from Perinucleolar Caps in Response to Genotoxic Stress (A) In order to induce nucleolar cap formation, HeLa cells were treated with 5 μg/ml ActD for up to 3 hr in the presence or absence of 40 μM PJ-34. Prior to formaldehyde fixation, cells were exposed to 2 mM H2O2 for 1 hr or 1 mM MMS for 2 hr and additionally pre-extracted for visualization of nucleolar caps. Immunostaining was performed with TAF15 and Fibrillarin antibodies. (B) Exemplary images taken from HCI analysis illustrating automated software-assisted detection of DAPI-stained nuclei and immunostained TAF15 caps. (C) Quantification of TAF15 cap intensities per nucleus of the conditions represented in (A) and additional conditions with ABT-888. Molecular Cell 2013 52, 272-285DOI: (10.1016/j.molcel.2013.08.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 PARylation-Dependent Accumulation of THRAP3 in Nuclear Speckles in Response to Genotoxic Stress (A) Stable THRAP3-GFP-expressing HeLa cells were treated with ActD and H2O2 and immunostained with rabbit polyclonal GFP and mouse monoclonal SC35 antibodies. (B) THRAP3-GFP cells were left untreated or treated with 5 mg/ml ActD for 3 hr in the presence or absence of 40 mM PJ-34. Prior to methanol fixation, cells were exposed to 2 mM H2O2 for the indicated time points and immunostained with mouse monoclonal GFP antibody and mouse monoclonal SC35 antibodies. (C) Quantification of THRAP3 speckle intensities per nucleus of the conditions represented in (A) and additional conditions with ABT-888. (D) Quantification of THRAP3 speckle intensities per nucleus of cells treated with siRNAs against ARTD1/PARP1 or ARTD2/PARP2 in the presence or absence of ActD and/or 2 mM H2O2. Molecular Cell 2013 52, 272-285DOI: (10.1016/j.molcel.2013.08.026) Copyright © 2013 Elsevier Inc. Terms and Conditions