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。在减数分裂过程中，程序化 双链断裂(DSB)允许同源染色体突触、交叉和分离 准确地说。众所周知，减数分裂过程中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是至关重要的。一个关键的DDR组件 是杂三聚体RAD9-Rad1-HUS1(9-1-1)络合物。在体细胞中，9-1-1复合体直接 招募DNA修复蛋白到损伤部位，并2)通过相互作用激活DDR信号 磷酸化的RAD9和TOPBP1的C末端尾巴，导致ATR激活，这反过来是至关重要的 DNA修复，细胞周期调节，以及整个基因组的维护。在减数分裂过程中，ATR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，9-1-1复合体在减数分裂过程中调节ATR信号的潜在机制仍然存在 人们对此知之甚少。增加复杂性的是，额外的替代9-1-1复合体 精母细胞中形成RAD9B和HUS1B。为了测试所有9-1-1复合体如何促进减数分裂，我们之前 获得睾丸特异的RAD1条件性基因敲除(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，ATR信号缺陷。由于RAD1的删除中断 钳形成，Rad1CKO模型没有区分信号依赖和 9-1-1复合体在减数分裂中的独立作用。为了具体了解9-1的生物学功能- 1介导的ATR激活，我们开发了具有丝氨酸到丙氨酸(SA)突变的功能分离突变体 在RAD9A和RAD9B的C末端尾部，破坏RAD9-TOPBP1的相互作用。这些老鼠突变体 是活的，而Rad9a或Rad9b的零突变会导致胚胎死亡。Rad9aSA/SA和Rad9bSA/SA 单突变体杂交产生Rad9aSA/SA/9bSA/SA双突变体，其中9-1-1/ATR信号转导 预计将被完全禁用。在目标1中，我将确定9-1-1依赖的ATR信号的影响 对生育和配子发生的干扰，包括它对程序化DSB修复的影响，配对 同源染色体和减数分裂前期的沉默。此外，我将比较减数分裂。 RAD9单或双突变体ATR信号中断对确定重叠或差异的影响 9-1-1复合体之间的功能。在目标2中，我将分析RAD9-TOPBP1如何 相互作用影响已知的和新的ATR底物在减数分裂中的磷酸化。新的911-和 ATR依赖的靶点将通过系统分析来自 Rad9aSA/SA/9bSA/SA、Rad1 CKO和ATR抑制剂处理(ATRI)小鼠。由于ATR信号的重要性 在减数分裂中，必须阐明减数分裂9-1-1/ATR信号网络如何实现高保真 配子产生，对人类生育能力和先天残疾有重要影响。