Phosphoproteomics of ATR signaling in mouse testes.

Jennie R Sims,Catalina Pereira,Vitor M Faça,Marcus B Smolka,Raimundo Freire,Paula E Cohen,Carolline Ascenção,Jumana Badar,Robert S Weiss,William Comstock,Gerardo A Arroyo-Martinez

doi:10.7554/elife.68648

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.

Highlights

Meiosis is a specialized cellular process whereby a single round of DNA replication is followed by two successive rounds of cell division to produce haploid gametes
ATR plays an essential role in spermatogenesis by promoting meiotic sex chromosome inactivation (MSCI), a process that is required for silencing of the X and Y chromosomes (Royo et al, 2010; Pacheco et al, 2018; Widger et al, 2018)
Our rationale was that phosphorylation impaired both by ATRi treatment and Rad1 cKO should reflect events that occur early in the ATR response and are specific to germ cells, overcoming limitations intrinsic to each of these experimental setups if analyzed in isolation

Summary

Introduction

Meiosis is a specialized cellular process whereby a single round of DNA replication is followed by two successive rounds of cell division to produce haploid gametes. Impairment of ATR activity results in insufficient MSCI and germ cell elimination at the mid-pachytene stage of prophase I (Widger et al, 2018; Royo et al, 2013; Turner, 2007; Menolfi et al, 2018; Fedoriw et al, 2015). ATR regulates the sex body localization of several other DNA damage response proteins such as MDC1 and BRCA1, resulting in MSCI (Royo et al, 2013; Turner et al, 2004; Ichijima et al, 2012). Loss of ATR protein in spermatocytes results in defects in DSB repair and chromosome synapsis, implying that ATR regulates several aspects of meiotic progression (Menolfi et al, 2018; Fedoriw et al, 2015; Widger et al, 2018). Despite the importance of ATR in meiosis, the mechanisms by which meiotic ATR signaling coordinates meiotic progression remain limited due to the complexity and interdependence of meiotic DNA repair, chromosome synapsis, and silencing of unsynapsed chromatin

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Results

Conclusion