Phosphoproteomics of ATR signaling in mouse testes
- Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, United States
- Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Brazil
- Department of Biomedical Sciences, Cornell University, United States
- Unidad de Investigación, Hospital Universitario de Canarias, Spain
- Instituto de Tecnologías Biomédicas, Universidad de La Laguna, Spain
- Universidad Fernando Pessoa Canarias, Spain
Figures
Figure 1 with 1 supplement
Experimental approach for identifying ATR-dependent phosphorylation events in meiosis.
(A) Schematics depicting the mechanism of ATR activation at a 5’ recessed DNA end via the 9-1-1 complex and TOPBP1, and strategies for chemical and genetic impairment of ATR signaling. (B) Whole, …
Figure 1—figure supplement 1
Imaging γH2AX on meiotic spreads from vehicle or ATRi-treated mice.
(A) Quantification of mean intensities for the ratio of γH2AX signal as depicted in Figure 1D, separated by individual animal replicates. γH2AX intensity is measured as described in Materials and …
Figure 2 with 4 supplements
ATR and RAD1-dependent signaling events in phosphoproteomic dataset corresponding to 4 hr ATRi treatment and Rad1 cKO.
(A) Scatter plot with assignment of phosphopeptides into quadrants delineated by dashed lines (‘bow-tie’ filter thresholds) and laying outside of a central region (‘center’ circle) comprised of …
Figure 2—figure supplement 1
Quadrant gene ontology of phosphoproteomic dataset from 4 hr ATRi treatment and Rad1 cKO.
(A) Scatter plot of phosphopeptides after ‘bow-tie’ filtering (dashed lines) and variability filtering (as described in Materials and methods) with colors indicating quadrant location for those …
Figure 2—figure supplement 2
ATR and RAD1-dependent signaling events in phosphoproteomic dataset of 2.5–3-day ATRi treatment and Rad1 cKO.
(A) Description of overall number of replicates and phosphopeptides identified. Full dataset can be found in Supplementary file 3. (B) Scatter plot with assignment of phosphopeptides into quadrants …
Figure 2—figure supplement 3
ClueGO analysis of ATR and RAD1-dependent events in Q2.
(A) Functional GO network generated using the ClueGO plugin for Cytoscape. Analysis of RAD1 and ATR-dependent phosphopeptides in Q2 after 4 hr treatment with ATR inhibitor. GO functional groups are …
Figure 2—figure supplement 4
MDC1 phosphorylation events after ATR inhibition.
Figure 3 with 2 supplements
RAD1 and ATR-dependent phosphorylation at the S/T-Q motif.
(A) Scatter plot highlighting all S/T-Q phosphorylation outside Q2 (dark gray) and S/T-Q phosphorylation inside Q2 (green). (B) Chord diagram of gene ontology of ATR and RAD1-dependent S/T-Q …
Figure 3—figure supplement 1
RAD1 and ATR-dependent phosphorylation at the S/T-Q motif in the 2.5–3-day ATRi vs. Rad1 cKO dataset.
(A) Selected set of proteins involved in chromatin modification and RNA metabolic processes with all identified phosphorylation sites ordered sequentially from the n-terminus to the c-terminus of …
Figure 3—figure supplement 2
Comparison of 2.5–3-day ATRi and 4 hr ATRi treatment Q2 datasets.
(A) Venn diagram of the number of Q2 peptides identified in the 2.5–3-day ATRi vs. Rad1 cKO dataset only (511 peptides, blue), the 4 hr ATRi vs. Rad1 cKO dataset only (261 peptides, green), and …
Figure 4 with 2 supplements
Senataxin (SETX) and RANBP3 localization in meiotic spreads after ATR inhibition.
(A) Immunofluorescence of meiotic chromosome spreads with SETX (green) and SYCP3 (red) from mice collected 4 hr after 50 mg/kg treatment with AZ20 or vehicle. (B) Quantification of pachytene spreads …
Figure 4—figure supplement 1
Effect of ATR inhibition on Senataxin (SETX) localization in meiotic spreads.
(A) Quantification of SETX signal at the sex body separated by individual mice. (B) Example images from vehicle or ATR inhibitor-treated mice collected 4 hr after 50 mg/kg treatment with AZ20 or …
Figure 4—figure supplement 2
Effect of ATR inhibition on RANBP3 localization in meiotic spreads.
(A) Quantification of RANBP3 intensity from meiotic spreads separated by animal. (B) Example spreads from mice collected 4 hr after 50 mg/kg treatment with AZ20 or vehicle. (C) Quantification of …
Figure 5 with 3 supplements
Enrichment of S/T-P-X-K phosphorylation motif in the set of ATR and RAD1-dependent signaling events.
(A) Bar graph depicting the count of Q2 phosphopeptides with the indicated amino acids at the +1 position. (B) Bar graph of the percentage of indicated phospho-motifs in the center (unchanged …
Figure 5—figure supplement 1
RAD1- and ATR-dependent signaling is enriched for phosphorylation events at the S/T-X-X-K motif.
(A) Heat map for the prevalence of amino acids at positions surrounding the phosphorylation sites (P: phosphorylation site position) comparing Q2 phosphopeptides to phosphopeptides found in the …
Figure 5—figure supplement 2
Effect of ATRi treatment on the localization of CDK2 in meiotic spreads.
(A) Quantification of autosomal core intensity of CDK2 as shown in Figure 5D, but separated by individual animal replicates. (B) Example pachynema images from vehicle or 4 hr ATRi-treated animals …
Figure 5—figure supplement 3
Histological and PCA analysis of ATRi treated mice.
(A) Hematoxylin and eosin stained testes tissue sections from vehicle and (B) 50 mg/kg AZ20 (ATRi)-treated mice. (C) Principal component analysis for experimental replicates and conditions. 4 hr …
Additional files
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Supplementary file 1
Complete phosphoproteomic dataset of the 4 hr ATRi vs. Rad1 cKO experiments.
Complete list of phosphopeptides identified in each biological replicate for mice treated with ATRi for 4 hr and the Rad1 cKO conditions. Column header definitions can be found in sheet 1. See Materials and methods for details on data processing and filtering. Phosphosites removed by filtering can be found in the last tab.
- https://cdn.elifesciences.org/articles/68648/elife-68648-supp1-v1.xlsx
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Supplementary file 2
Gene ontology STRING analysis output.
Gene ontology information from STRING-db for subsets of data from the 4 hr or 2.5–3-day ATRi treatment combined to Rad1 cKO. Each quadrant or type of filter applied is indicated on the tab name.
- https://cdn.elifesciences.org/articles/68648/elife-68648-supp2-v1.xlsx
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Supplementary file 3
Complete phosphoproteomic database of the 2.5–3-day ATRi vs. Rad1 cKO experiments.
Complete list of phosphopeptides identified in each biological replicate for mice treated with ATRi 2.5 and 3 days hours and the Rad1 cKO conditions. Rad1 cKO phosphopeptides are the same presented in Supplementary file 1. Column header definitions can be found in sheet 1. See Materials and methods for details on data processing and filtering. Phosphosites removed by filtering can be found in the last tab.
- https://cdn.elifesciences.org/articles/68648/elife-68648-supp3-v1.xlsx
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Supplementary file 4
Phosphoproteomic database comparison of Q2 4 hr ATRi and 2.5–3-day ATRi.
Comparison of the Q2 phosphopeptides found in the 4 hr and/or 2.5–3-day ATRi treatment conditions. Data is separated into Q2 sites exclusively found in the 4 hr ATRi treatment, 2.5–3 hr ATRi treatment or sites common between both datasets, separated into different sheets.
- https://cdn.elifesciences.org/articles/68648/elife-68648-supp4-v1.xlsx
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Supplementary file 5
Gene ontology chord analysis output.
Gene ontology output from chord diagrams. See Materials and methods for more details.
- https://cdn.elifesciences.org/articles/68648/elife-68648-supp5-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68648/elife-68648-transrepform1-v1.pdf
