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Activation of the ATR kinase by the RPA-binding protein ETAA1

Abstract

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing’s tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate that parallel TopBP1- and ETAA1-mediated pathways underlie ATR activation and that their combined action is essential for coping with replication stress.

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Figure 1: Systematic profiling of DSB-containing chromatin reveals ETAA1 as a potential DDR factor.
Figure 2: ETAA1 is recruited to DNA damage sites through interaction with RPA.
Figure 3: ETAA1 contains an ATR-activating domain (AAD).
Figure 4: ETAA1 promotes cell survival and ATR signalling after replication stress.
Figure 5: ETAA1 promotes DNA replication integrity during unperturbed and stressful conditions.
Figure 6: ETAA1 and TopBP1 promote parallel, but independent, pathways of ATR activation.
Figure 7: ETAA1 and TopBP1 both contribute to ATR signalling in cancer cell lines.

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Acknowledgements

We thank D. Cortez and A. Kumagai for providing reagents. We thank K. Mayr, I. Paron and G. Sowa for mass spectrometry support and M. Steger and B. Splettstösser (all from Max Planck Institute of Biochemistry) for experimental advice. This work was supported by grants from The Novo Nordisk Foundation (grants no. NNF14CC0001 and NNF12OC0002114), European Research Council (ERC, grant agreement no. 616236 (DDRegulation)), The Danish Cancer Society, The Danish Council for Independent Research, Danish National Research Foundation (grant no. DNRF115) and Center for Integrated Protein Science Munich (CIPSM).

Author information

Authors and Affiliations

Authors

Contributions

P.H. and N.M. conceived the study. P.H., S.H., M.A.X.T. and T.H. designed and performed cell-, biochemistry- and imaging-based experiments and analysed the data, under the supervision of N.M. M.R. designed and performed mass spectrometry experiments and analysed the data, under the supervision of M.M. L.I.T. provided help and support with quantitative image analysis and time-lapse microscopy. S.B.-J. co-supervised the study. N.M. wrote the manuscript with inputs from P.H. and M.R. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Markus Räschle or Niels Mailand.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Systematic profiling of DSB-containing chromatin by CHROMASS.

(a) Etaa1 sequence coverage in CHROMASS experiments. Identified peptides are indicated by red bars above the amino acid sequence. (b) Profile plots for the recruitment of Etaa1 and other indicated factors to DSB-containing chromatin at different time points. The z-scored log2 LFQ intensity (mean of three replicates) was plotted against time. Note that the recruitment profile of Etaa1 closely follows that of the RPA complex (RPA1-3). (c) Cluster analysis. The mean averaged intensity profile of RPA (Rfa1, Rfa2 and Rfa3, shown in dark green in Supplementary Fig. 1b) was used to calculate distances for proteins (2668 in total) that had at least 50% valid values. Proteins were ranked according to increasing distances. Analysis was done with the Profile Plot function implemented in Perseus. (d) Undamaged chromatin was incubated in HSS Xenopus egg extract to license chromatin for replication. DNA replication was initiated by addition of NPE Xenopus egg extract. Aphidicolin was added to NPE to inhibit DNA polymerases, thereby triggering uncoupling of replicative helicase and polymerase movements (see right panel). The volcano plot shows the mean difference of the protein intensity relative to reactions containing the replication inhibitor geminin plotted against the P value. The graph was replotted from primary data described in31.

Supplementary Figure 2 ETAA1 accumulates at DNA damage sites through binding to RPA.

(a) Immunoblot analysis of U2OS/GFP-ETAA1 cells transfected with indicated siRNAs. See also Fig. 2a. (b) U2OS cells transiently transfected with constructs encoding indicated GFP-ETAA1 fragments were subjected to immunoprecipitation (IP) with GFP-TRAP beads followed by immunoblotting with GFP and RPA2 antibodies. (c) Alignment of RPA1- and RPA2-binding motifs in vertebrate ETAA1 proteins. (d) U2OS cell lines stably expressing indicated WT and mutant GFP-ETAA1 constructs were subjected to laser microirradiation, fixed 1 h later and immunostained with γH2AX antibody. RQ(902-3)AA, R902A, Q903A. (e) Immunoblot analysis of U2OS cell lines stably expressing indicated GFP-ETAA1 proteins. (f) U2OS cell lines stably expressing indicated GFP-ETAA1 proteins were fixed and immunostained with RPA1 antibody. All scale bars, 10 μm. Data are representative of three (b,d) and two (a,e,f) independent experiments with similar results. Uncropped blots (a,b,e) are shown in Supplementary Fig. 8.

Supplementary Figure 3 Characterization of an ATR-activating domain (AAD) in ETAA1.

(a) Location and sequence conservation of the ETAA1 AAD. Highly conserved residues are highlighted (blue). Residues mutated to alanine in order to generate the AADmut allele are indicated in red. (b) Overview of GFP-ETAA1 fragments used and their ability to induce pan-nuclear γ-H2AX formation when overexpressed in U2OS cells. (c) U2OS cells stably expressing GFP-ETAA1-AAD-ERT2 were exposed to 4-OHT for various times and immunostained with γH2AX antibody. Mean γH2AX intensity per nucleus was determined by quantitative image analysis (n = 3000 cells per condition). (d) U2OS cells transfected with GFP empty vector (EV) or constructs encoding GFP-ETAA1-AAD or GFP-TopBP1-AAD were fixed and stained with γH2AX antibody. Cells were gated according to mean GFP intensity per nucleus (shades of green). γH2AX signal per nucleus was determined by quantitative image analysis (1,000 cells analyzed per condition) (bottom). (e) Quantitative image analysis of U2OS/GFP-ETAA1-AAD-ERT2 cells treated with 4-OHT for 2 h and co-immunostained with antibodies to RPA1 and γH2AX (5,000 cells analyzed per condition). The color of each dot represents mean γH2AX intensity per nucleus, ranging from low (grey) to high (red) (left). Bar chart shows the mean γH2AX and RPA1 signals per nucleus normalized to mock control (right). (f) U2OS/GFP-ETAA1-AAD-ERT2 cells left untreated or induced to express the transgene by addition of Doxycycline (DOX) were subjected to GFP immunoprecipitation (IP) followed by immunoblotting with GFP and ATR antibodies. (g) HCT116 cells were exposed to HU (3 h) or CPT (2 h) in the presence or absence of ATR inhibitor (ATRi) and analyzed by immunoblotting with indicated antibodies. Asterisk denotes an unspecific band recognized by the ETAA1 antibody. (h) Setup of phospho-proteomics experiments in Fig. 3i. Cells were subjected to SILAC labeling with light medium (L; Lys0, Arg0) or heavy medium (H; Lys8, Arg10) and treated with 4-OHT and/or ATR inhibitor (ATRi) for 1 h as indicated. i. Venn diagrams showing overlap between regulated phosphorylation sites (N denotes total number) in mass spectrometry experiments in h. Data are representative of three (e,f,g) and two (c,d) independent experiments with similar results. Uncropped blots (f,g) are shown in Supplementary Fig. 8.

Supplementary Figure 4 ETAA1 promotes signaling and cell survival in response to replication stress.

(a) Quantitative image analysis of asynchronously growing HCT116 WT and ETAA1Δcell lines labelled with EdU and stained with DAPI (n = 2000 cells per condition). Proportion of cells in different cell cycle phases (blue: G1 phase; red: S phase; green: G2/M phase) is indicated. (b) As in a, but using HeLa WT and ETAA1Δ cell lines 2000 cells analyzed per condition). (c) Immunoblot analysis of parental HeLa (WT) cells and two independent, derivative ETAA1 knockout lines (ETAA1Δ) left untreated or exposed to CPT for 90 min. (d) HCT116 WT and ETAA1Δ cell lines were treated with HU for the indicated times and analyzed by immunoblotting. Asterisk demarcates an unspecific band detected by the ETAA1 antibody. (e) Clonogenic survival assay, using HCT116 WT and ETAA1Δ-3 cell lines subjected to indicated doses of HU for 24 h. (f) As in e, except cells were treated with indicated concentrations of CPT for 24 h. (g) Clonogenic survival assay, using HCT116 WT, ETAA1Δ-1 and ETAA1Δ-3 cell lines subjected to indicated doses of Etoposide for 1 h. (h) Clonogenic survival of HCT116 cells transfected with non-targeting control (CTRL) or ETAA1 siRNAs and exposed to indicated doses of HU for 24 h. (i) As in h, except that cells were exposed to indicated doses of CPT for 24 h. Error bars in ei are mean ± s.e.m.; n = 3 independent experiments. (j) Immunoblot analysis of cells used for the experiments in h and i. (k) Immunoblot analysis of HCT116 WT, ETAA1Δand ETAA1Δ cell lines stably reconstituted with indicated forms of GFP-ETAA1. (l) Cell cycle profiles of the cell lines in k, determined as in a (2000 cells analyzed per condition). Data are representative of three (c,d,j) and two (a,b,k,l) independent experiments with similar results. Uncropped blots (c,d,j,k) are shown in Supplementary Fig. 8.

Supplementary Figure 5 ETAA1 promotes the integrity of normal DNA replication.

(a) DNA fiber analysis of HeLa WT and ETAA1Δ cells labeled with CldU (20 min) followed by IdU (20 min). Fork speeds were calculated as length of labeled track divided by pulse time (200 fibers analyzed per condition). Red bars denote median fork speed. (b) Fork symmetry was calculated as the percentage of shorter divided by longer tracks from (a) (25 bidirectional forks analyzed per condition). Concordance is 100%, representing fully bidirectional replication and equal rates of elongation for both daughter forks. Red bars denote median fork symmetry c. As in a, except that the proportion of new origins (IdU-only tracks) was determined (200 fibers analyzed per condition). (d) Immunoblot analysis of HCT116 cells transfected with non-targeting control (CTRL) or ETAA1 siRNAs. (e) HeLa cells transfected with indicated siRNAs were labeled with EdU, fixed, and processed for γH2AX and EdU staining. Cells were then analyzed by quantitative image analysis (n = 3000 cells per condition). Cells were gated into G1 and S/G2 cell populations based on EdU and DAPI intensities, and analyzed for mean γH2AX signal per nucleus. Box plot shows median, upper and lower quartiles, 95% values (whiskers) and outliers. (f) As in e, but using untransfected HeLa WT and ETAA1Δ cell lines (n = 3000 cells per condition) Box plot shows median, upper and lower quartiles, 95% values (whiskers) and outliers. Data are representative of three (e) and two (d,f) independent experiments with similar results. Fiber data (ac) represents a single technical repeat but represents a biological repeat of data in Fig. 5a–c. Uncropped blots (d) are shown in Supplementary Fig. 8.

Supplementary Figure 6 ETAA1 and TopBP1 promote ATR activation through parallel, but independent, pathways.

(a) Quantitative image analysis of U2OS/GFP-ETAA1-AAD-ERT2 cells transfected with indicated siRNAs, treated with 4-OHT for 1 h, and immunostained with γH2AX antibody (mean ± s.d.; n = 3 independent experiments).b. As in (a), but using indicated siRNAs (n = 3000 cells per condition). (c) Immunoblot analysis of U2OS/GFP-ETAA1-AAD-ERT2 cells transfected with siRNAs used in a and b. (d) HCT116 WT or ETAA1Δ cells transiently transfected with plasmids encoding GFP-TopBP1-AAD WT or a W1145R point mutant (W R) unable to stimulate ATR activity were fixed, immunostained with γH2AX antibody and analyzed for GFP and γH2AX signal intensity by quantitative image analysis. Mean γH2AX signal per nucleus of GFP-positive cells (green; 350 cells analyzed per condition) are shown in the bar chart to the right. (e) Immunoblot analysis of HeLa WT and ETAA1Δ cells transfected with control (−) or TopBP1 siRNAs for 72 h. Asterisk demarcates an unspecific band detected by the ETAA1 antibody. (f) Immunoblot analysis of HCT116 cells transfected with indicated combinations of non-targeting control (CTRL), TopBP1 and ETAA1 siRNAs. (g) Immunoblot analysis of HCT116 cells treated with ATR inhibitor (ATRi) for the indicated times. (h) Cells in f were stained with DAPI and analyzed by quantitative image analysis to determine their cell cycle profiles and the proportion of sub-G1 DNA content nuclei (pink) (3000 nuclei analyzed per condition). (i) HCT116 ETAA1Δ cells transfected with non-targeting control (CTRL) or TopBP1 siRNAs were co-immunostained with γH2AX and H3 phospho-Ser10 antibodies. Scale bar, 10 μm. (j) HCT116 ETAA1Δ-3 cells were treated with non-targeting control (CTRL) or TopBP1 siRNAs and analyzed as in h. Eight h after siRNA transfection, Z-VAD-FMK (10 μM) was added to inhibit apoptosis for the duration of the experiment. Proportion of nuclei with sub-G1 DNA content (pink) is indicated (3000 cells analyzed per condition). Data are representative of three (f,g) and two (be,hj) independent experiments with similar results. Uncropped blots (c,eg) are shown in Supplementary Fig. 8.

Supplementary Figure 7 Characterization of ETAA1 in HeLa and U2OS cells.

(a) qPCR analysis of ETAA1 mRNA levels in different cell lines relative to HCT116 cells. Primers to GAPDH were used as a control for normalization (mean ± s.e.m.; n = 3 independent experiments).b. As in a, but using primers to ubiquitin as a control for normalization (mean ± s.e.m.; n = 3 independent experiments). (c) U2OS and HeLa were treated with Cycloheximide (CHX) for the indicated times and analyzed by immunoblotting to assess the half-life of ETAA1. Cyclin F was as a positive control for the effect of CHX. Asterisk denotes an unspecific band recognized by the ETAA1 antibody. (d) HeLa cells transfected with indicated siRNAs were treated with HU and/or ATR inhibitor (ATRi), stained with RPA1 and γH2AX antibodies and subjected to quantitative image analysis (3500 cells analyzed per condition). The contribution of ATR to the γH2AX signal, based on the ATRi-treated sample, is indicated in blue. Cells displaying maximal HU-induced RPA chromatin loading accompanied by ATM-dependent but ATR-independent H2AX hyperphosphorylation reflecting fork breakage42 are indicated in pink. (e) Quantification (mean ± s.e.m.) of γH2AX signal per nucleus of the ATR-gated population (blue) in d (n = 1500 cells per condition). ( f) Immunoblot analysis of the experiment in (c). Data are representative of two (cf) independent experiments with similar results. Source data (a,b) are provided in Supplementary Table 5. Uncropped blots (c,f) are shown in Supplementary Fig. 8.

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Representative example of mitotic progression in cells lacking TopBP1.

HCT116 WT cells stably reconstituted with GFP-H2B were transfected with TopBP1 siRNA and monitored by live cell fluorescence microscopy at 24–60 h after siRNA transfection. Video shows representative examples of mitosis under these conditions. (AVI 16529 kb)

Representative example of mitotic progression in cells lacking ETAA1 and TopBP1.

HCT116 ETAA1Δ-3 cells stably reconstituted with GFP-H2B were transfected with TopBP1 siRNA and followed by live cell fluorescence microscopy at 24–60 h after siRNA transfection. Video shows representative examples of mitotic errors leading to nuclear fragmentation under these conditions. (AVI 47556 kb)

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Haahr, P., Hoffmann, S., Tollenaere, M. et al. Activation of the ATR kinase by the RPA-binding protein ETAA1. Nat Cell Biol 18, 1196–1207 (2016). https://doi.org/10.1038/ncb3422

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