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.

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here, we report that testis-specific disruption of RAD1 in mice resulted in impaired DSB repair, germ cell depletion, and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss in meiocytes also caused severe defects in homolog synapsis, impaired phosphorylation of ATR targets such as H2AX, CHK1, and HORMAD2, and compromised meiotic sex chromosome inactivation. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

Every year, approximately half a million people die due to breast cancer (BC), comprising 16% of the incidence of all cancer occurrences in females. It is the most prevalent invasive cancer among women around the world. In several studies, researchers have suggested the presence of certain chemokines associated with cancer. The chemokine receptor CXCR4, a G‐protein coupled receptor, has a role in trafficking, cell activation, and differentiation in non‐malignant. It has been shown that CXCR4 is overexpressed in cancerous tissues compared to non‐malignant cells. This overexpression has led to an increase in proliferation, migration, and angiogenesis of malignant cells. Tissues with higher expression of the CXCR4 specific ligand CXCL12, are more likely to metastasis to tissue expressing CXCR4. These target tissues include lungs, bone marrow, and lymph nodes. Due to the impact of this disease, there is a need of developing new therapies or adjuvants to target BC. For example, resveratrol is a natural product and a polyphenol derived from grapes, wine, and nuts, among others. Recent studies found that it plays a role in apoptosis, inflammation, and neovascularization. We hypothesized that resveratrol will decrease the expression of CXCR4 in a dose and time‐dependent manner, leading to a decrease in the migration and invasion cellular processes. Using western blot analysis of HS578T (Human Breast Carcinoma) compared with CCD1074Sk (Non‐malignant Human Breast Tissue) cell lines we studied the effects of resveratrol at the CXCR4 protein level and invasion markers involved in metastatic processes. Migration and invasion were assessed using a wound healing assay and matrigel invasion assay, respectively. The preliminary data shows that as the concentration (25, 50, 100, 200μM) increases the cell migration and invasion decreases compared to our control vehicle (EtOH). In addition, we observed a decrease in the expression of pro‐migration and invasion proteins such as N‐cadherin and Cathepsin‐b. This study suggests an effect of resveratrol in cell migration and invasion leading to a decrease of the in‐vitro cell metastatic potential. Currently we are evaluating CXCR4 at the protein expression level to complete our data.Support or Funding InformationThis work was supported by UPR PRISE Program NIH‐NIGMS #2R25GM096955This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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