ATR signaling activation by the 9-1-1 complexes during mammalian meiosis

哺乳动物减数分裂期间 9-1-1 复合物激活 ATR 信号

• 批准号: 10680036
• 负责人: Gerardo Antonio Arroyo Martinez
• 金额: $ 4.42万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680036
• 关键词: Alanine; Biological Process; C-terminal; CSPG6 gene; Cell Cycle Regulation; Cell physiology; Chromosome Pairing; Chromosome Structures; Chromosomes; Closure by clamp; Complex; DNA Damage; DNA Repair; DNA Repair Gene; Data; Defect; Disabled Persons; Double Strand Break Repair; Embryo; Evaluation; Event; Fertility; Fostering; Gametogenesis; Genetic Crossing Over; Genome; Genome Stability; Genomic Instability; Germ Cells; Haploidy; Histologic; Homologous Gene; Human; Impairment; Infertility; Knockout Mice; Knowledge; Maintenance; Mediating; Meiosis; Meiotic Prophase I; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Process; Production; RAD9A gene; Regulation; Reporting; Reproductive Health; Role; SETX gene; Serine; Sex Chromosomes; Signal Transduction; Site; Somatic Cell; Spermatocytes; Synapses; Synaptonemal Complex; TOPBP1 Gene; Tail; Testing; Testis; conditional knockout; disability; experimental study; homologous recombination; improved; inhibitor; mutant; novel; null mutation; paralogous gene; phosphoproteomics; prevent; recruit; repaired; response; segregation; sensor

Meiosis is a highly regulated cellular process for generating haploid gametes. During meiosis, programmed double-strand breaks (DSB) allow homologous chromosomes to synapse, crossover, and segregate accurately. Chromosomal errors during meiosis are known to result in infertility or congenital disabilities. Consequently, DNA damage response (DDR) mechanisms are crucial during meiosis. A key DDR component is the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) complex. In somatic cells, the 9-1-1 complex 1) directly recruits DNA repair proteins to damage sites and 2) activates DDR signaling via interactions between the phosphorylated C-terminal tail of RAD9 and TOPBP1, resulting in ATR activation, which in turn is crucial for DNA repair, cell cycle regulation, and overall genome maintenance. During meiosis, ATR is known to promote homologous recombination and is a critical player in promoting meiotic silencing at unsynapsed regions. However, the underlying mechanism of the 9-1-1 complex in regulating ATR signaling during meiosis remains poorly understood. Adding to the complexity, additional alternative 9-1-1 complexes involving the paralogs RAD9B and HUS1B form in spermatocytes. To test how all 9-1-1 complexes promote meiosis, we previously generated testis-specific Rad1 conditional knock-out (CKO) mice and observed severe asynapsis, compromised DSB repair, impaired meiotic silencing, and ATR signaling defects. Since Rad1 deletion disrupts clamp formation, the Rad1 CKO model does not differentiate between the signaling-dependent and independent roles of the 9-1-1 complexes in meiosis. To specifically understand the biological functions of 9-1- 1 mediated ATR activation, we developed separation-of-function mutants with serine-to-alanine (SA) mutations in the C-terminal tail of RAD9A and RAD9B that disrupt RAD9-TOPBP1 interactions. These mouse mutants were viable, whereas null mutations in Rad9a or Rad9b cause embryonic lethality. Rad9aSA/SA and Rad9bSA/SA single mutants were inter-crossed to generate Rad9aSA/SA/9bSA/SA double mutants in which 9-1-1/ATR signaling is predicted to be fully disabled. In Aim 1, I will determine the effects of 9-1-1-dependent ATR signaling disruption on fertility and gametogenesis, including its impact on the repair of programmed DSBs, pairing of homologous chromosomes, and meiotic silencing during prophase I. In addition, I will compare the meiotic effects of ATR signaling disruption in Rad9 single or double mutants to determine overlapping or differential functions between the canonical and alternative 9-1-1 complexes. In Aim 2, I will analyze how RAD9-TOPBP1 interactions influence the phosphorylation of known and novel ATR substrates during meiosis. New 911- and ATR- dependent targets will be identified by systematic analysis of whole testes phosphoproteomes from Rad9aSA/SA/9bSA/SA, Rad1 CKO and ATR inhibitor-treated (ATRi) mice. Due to the importance of ATR signaling in meiosis, it is imperative to shed light on how the meiotic 9-1-1/ATR signaling network enables high-fidelity gamete production, with important implications for human fertility and congenital disabilities.

减数分裂是产生单倍体配子的高度调控的细胞过程。在减数分裂期间，程序化 双链断裂 (DSB) 允许同源染色体突触、交叉和分离 准确。已知减数分裂期间的染色体错误会导致不孕或先天性残疾。 因此，DNA 损伤反应 (DDR) 机制在减数分裂过程中至关重要。 DDR 的关键组件 是异源三聚体 RAD9-RAD1-HUS1 (9-1-1) 复合物。在体细胞中，9-1-1 复合体 1) 直接 招募 DNA 修复蛋白到损伤位点，2) 通过 DNA 修复蛋白之间的相互作用激活 DDR 信号传导 RAD9 和 TOPBP1 的 C 末端尾部磷酸化，导致 ATR 激活，这反过来对于 DNA 修复、细胞周期调节和整体基因组维护。在减数分裂期间，ATR 会促进 同源重组，是促进非突触区域减数分裂沉默的关键因素。 然而，减数分裂期间 9-1-1 复合物调节 ATR 信号传导的潜在机制仍然存在 不太了解。涉及旁系同源物的其他替代 9-1-1 复合物增加了复杂性 RAD9B 和 HUS1B 在精母细胞中形成。为了测试所有 9-1-1 复合物如何促进减数分裂，我们之前 生成睾丸特异性 Rad1 条件敲除 (CKO) 小鼠并观察到严重的突触， DSB 修复受损、减数分裂沉默受损和 ATR 信号传导缺陷。由于 Rad1 删除会破坏 钳形成，Rad1 CKO 模型不区分信号依赖型和 9-1-1复合物在减数分裂中的独立作用。具体了解9-1-的生物学功能 1 介导 ATR 激活，我们开发了具有丝氨酸至丙氨酸 (SA) 突变的功能分离突变体 位于 RAD9A 和 RAD9B 的 C 末端尾部，破坏 RAD9-TOPBP1 相互作用。这些小鼠突变体 是可行的，而 Rad9a 或 Rad9b 的无效突变会导致胚胎致死。 Rad9aSA/SA 和 Rad9bSA/SA 单突变体交叉产生 Rad9aSA/SA/9bSA/SA 双突变体，其中 9-1-1/ATR 信号传导 预计将完全瘫痪。在目标 1 中，我将确定 9-1-1 依赖性 ATR 信号传导的影响 对生育力和配子发生的破坏，包括其对程序化 DSB 修复、配对的影响 同源染色体，以及前期 I 期间的减数分裂沉默。此外，我将比较减数分裂 Rad9 单突变体或双突变体中 ATR 信号破坏的影响以确定重叠或差异 介于规范 9-1-1 复合体和替代 9-1-1 复合体之间的功能。在目标 2 中，我将分析 RAD9-TOPBP1 如何 相互作用影响减数分裂过程中已知和新型 ATR 底物的磷酸化。新 911- 和 ATR 依赖性靶标将通过对来自以下来源的整个睾丸磷酸化蛋白质组进行系统分析来确定： Rad9aSA/SA/9bSA/SA、Rad1 CKO 和 ATR 抑制剂治疗的 (ATRi) 小鼠。由于 ATR 信号的重要性 在减数分裂中，有必要阐明减数分裂 9-1-1/ATR 信号网络如何实现高保真 配子的产生，对人类生育能力和先天性残疾具有重要影响。