Phosphoproteomics of ATR signaling in mouse testes

Abstract

The phosphatidylinositol 3′ kinase (PI3K)‐related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.

Active DNA damage response signaling initiates and maintains meiotic sex chromosome inactivation

“…We found that the γH2AX domain disappeared from the XY chromatin in H1T-positive mid-pachytene spermatocytes following treatment with AZ20, but not in testes from untreated mice (Fig. 8b), consistent with another independent study using the same condition 59 . To examine the maintenance of MSCI in this mouse model, we prepared chromosome spreads using cell suspensions.…”

“…A recent study with AZ20, which was originally developed for chemotherapy, used an in vivo mouse model and a testicular organ culture model to show that ATR is required to complete meiotic recombination 20 . Another study used AZ20 for phosphoproteomics of ATR signaling in mouse testes using an in vivo mouse model 59 . In our study, we instead focused on treating mouse spermatocytes ex vivo with two different ATR inhibitors to specifically address the function of ATR in MSCI.…”

“…ATR inhibitor treatment by gavage was performed as described 20,59 . Briefly, the AZ20 compound (MedChemExpress) was dissolved in 10% DMSO/40% propylene glycol/50% water and was administered orally to adult B6 males at a single dose of 50 mg/kg.…”

The DNA helicase FANCJ (BRIP1) functions in Double Strand Break repair processing, but not crossover formation during Prophase I of meiosis in male mice

“…Proteins eluted from anti-HA beads were processed for mass spectrometry analysis following a previously described protocol that includes reduction, alkylation, precipitation, and digestion by trypsin over-night 88 . Peptides were then desalted, dried 89 and labelled with TMT following a protocol described previously 90 . Briefly, the dried peptides were resuspended in 50 mM HEPES, and labeled with 100 µg of TMT sixplex Isobaric Label Reagents (Thermo Scientific) using 3 TMT channels for each sample ( Wild-type and FANCJ-HA ).…”

Exploring the cardiac ECM during fibrosis: A new era with next-gen proteomics

“…These challenges can only be overcome by utilizing MS-based proteomics ( Humphrey et al, 2018 ; Savage and Zhang, 2020 ; Liu et al, 2021 ). The field of phosphoproteomics has emerged greatly in the last decade ( Lundby et al, 2013 ; Sharma et al, 2014 ; Alam et al, 2015 ; Riley and Coon, 2016 ; Steger et al, 2016 ; Tan et al, 2017 ; Kuzmanov et al, 2020a ; Wu et al, 2020 ; Sims et al, 2022 ). It has contributed significantly to biological research.…”