Investigating conserved mechanisms that orchestrate the prophase to metaphase transition during meiosis I

研究减数分裂 I 期间协调前期到中期转变的保守机制

• 批准号: 10677686
• 负责人: Ian Wolff
• 金额: $ 7.18万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10677686
• 关键词: Acute; Adaptor Signaling Protein; Address; Aneuploidy; Animals; Auxins; Binding Proteins; Biochemical; Biochemistry; Biological Assay; CDC2 gene; CUL1 gene; Caenorhabditis elegans; Cell Cycle; Cell Cycle Progression; Cell Nucleus; Cells; Chromatin; Chromosome Pairing; Chromosome Segregation; Chromosomes; Complex; Conceptions; Coupled; Cullin Proteins; Cyclin B; Cyclins; Data; Defect; Development; Development Plans; Diploidy; Embryo; Ensure; Enzymes; Eukaryota; Event; F Box Domain; Family; Female; Gametogenesis; Genetic; Genetic Crossing Over; Genetic Materials; Germ Cells; Germ Lines; Haploidy; Homologous Gene; House mice; Human; Individual; Investigation; Knockout Mice; Knowledge; Laboratories; Laboratory mice; Lead; Licensing; Licensing Factor; Life; Lighting; Maturation-Promoting Factor; Mediating; Meiosis; Meiotic Prophase I; Metaphase; Microscopy; Mitosis; Mitotic; Modeling; Molecular; Mus; Mutant Strains Mice; Nematoda; Oogenesis; Organism; Orthologous Gene; Phase Transition; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Positioning Attribute; Pregnancy Complications; Process; Prophase; Proteins; Publishing; RNA Interference; RNA interference screen; Regulation; Reproduction; Reproductive Biology; Research; Resolution; Role; Signal Transduction; Spermatocytes; System; Techniques; Testing; Testis; Training; Ubiquitin-Conjugating Enzymes; Ubiquitination; Universities; Veterinary Medicine; Visualization; Work; career development; college; competence factor; egg; experimental study; genetic information; inhibitor; male; member; model organism; mutant; precursor cell; prevent; segregation; sperm cell; three dimensional structure; ubiquitin-protein ligase

Sexually reproducing organisms generate gametes through meiosis, the process by which the genetic material of the cell is halved to form a haploid sperm or egg. Although essential for all animal life, meiosis is strikingly error-prone, with an estimated 5-25% of all human conceptions resulting in an aneuploid embryo, often leading to severe developmental defects and pregnancy complications that effect millions of individuals every year. Therefore, it is critical to investigate the core molecular mechanisms that define progression through meiosis to further understand what checkpoints may exist to sense errors. This proposal investigates the control of meiotic progression using two powerful meiotic models, the lab mouse Mus musculus and the nematode Caenorhabditis elegans. Meiosis I proceeds through pairing, synapsis, and crossing over (CO) of homologous chromosomes during prophase, and only once these events have occurred correctly can homologs align appropriately on the meiotic spindle and then segregate equally at the first meiotic division. However, it is unclear how successful CO formation is sensed by cell cycle machinery to progress meiosis into M-phase. Proper CO formation requires orthologous cyclin-like domain containing proteins CNTD1, in mouse spermatocytes, and COSA-1, in C. elegans oogenesis. Interestingly, CNTD1 interacts with CDC34, a ubiquitin E2 conjugating enzyme that acts with the Skp1-Cullin-F-Box (SCF) family of E3 ubiquitin ligases. A known target of CDC34 in mitotic systems is the cell cycle M-phase inhibitor, WEE1. In CNTD1 knockout mouse spermatocytes, CDC34 is absent from nuclei and WEE1 persists aberrantly through until the end of meiotic prophase, suggesting a role for CNTD1-promoted crossover maturation in WEE1 degradation and resulting progression into M-phase. This leads to the hypothesis that progression from meiotic prophase I to M-phase I is licensed through direct WEE1 ubiquitination by SCFCDC34 in a crossover-dependent manner that is conserved across eukaryotes. This proposal presents two specific aims: 1) Is SCFCDC34-mediated ubiquitination of WEE1 critical for progression from meiotic prophase I to M-phase I? WEE1 ubiquitination and WEE1/CDC34 chromatin localization will be directly tested in both wild type and crossover deficient mutant mice. An assay will be developed to directly visualize the germ line abundance of the WEE1 ortholog in C. elegans, WEE-1.3, to test the role of WEE-1.3 in C. elegans meiotic progression. 2) What is the biochemical mechanism of SCFCDC34 mediated WEE1 ubiquitination? The molecular components of the SCFCDC34 complex will be elucidated in mouse spermatocytes through mutant analysis of putative complex members, and in C. elegans through a RNAi screen. This work will be performed in the lab of Dr. Paula Cohen in the Cornell University College of Veterinary Medicine, providing extensive training in mammalian reproductive biology, specifically in the early meiotic events during gametogenesis. This research strategy coupled with the applicant’s training and career development plans will provide a unique approach towards investigation of meiosis through using complementary model organisms.

有性繁殖生物通过减数分裂产生配子，遗传物质通过减数分裂产生配子 减半形成单倍体精子或卵子。尽管减数分裂对所有动物都是必不可少的，但减数分裂是惊人的 容易出错，估计有5%-25%的人类受孕导致非整倍体胚胎，通常导致 严重的发育缺陷和妊娠并发症，每年影响数百万人。 因此，研究决定减数分裂进程的核心分子机制是至关重要的。 进一步了解可能存在哪些检查点来检测错误。这项提案调查了对 用两种强大的减数分裂模型--小鼠和线虫--进行减数分裂 秀丽隐杆线虫。减数分裂I通过同源基因的配对、突触和交换(CO)进行。 染色体处于早期阶段，只有当这些事件正确发生时，同源基因才能对齐 在减数分裂纺锤体上适当分离，然后在第一次减数分裂时均匀分离。然而，目前还不清楚 细胞周期机制如何感知CO的成功形成以使减数分裂进入M期。适当的CO 在小鼠精母细胞中，形成需要含有同源周期蛋白样域的蛋白CNTD1，以及 COSA-1，在线虫卵子发生中。有趣的是，CNTD1与CDC34相互作用，CDC34是泛素E2的结合体 与E3泛素连接酶的Skp1-cullin-F-Box(SCF)家族作用的酶。CDC34的一个已知目标是 有丝分裂系统是细胞周期的M期抑制因子，WEE1。在CNTD1基因敲除的小鼠精母细胞中，CDC34是 没有细胞核，WEE1异常地持续到减数分裂前期结束，这表明WEE1在减数分裂前期 对于CNTD1促进的WEE1降解和由此进展到M期的交叉成熟。这 导致假设从减数分裂前期I到M期I的进展是通过直接 SCFCDC34以交叉依赖的方式泛素化，这在真核生物中是保守的。 这一提议提出了两个具体目标：1)SCFCDC34介导的WEE1泛素化对进展至关重要 从减数分裂前期I到M期I？Wee1泛素化和WEE1/CDC34染色质定位 在野生型和杂交缺陷突变小鼠身上进行了直接测试。一种化验方法将被开发成直接 可视化线虫WEE1同源基因在线虫WEE-1.3中的胚系丰度，以测试WEE-1.3在 线虫减数分裂进程。2)SCFCDC34介导WEE1的生化机制是什么 泛素化？SCFCDC34复合体的分子组成将在小鼠精母细胞中得到阐明 通过对推测的复合体成员的突变分析，以及通过RNAi筛选在线虫中。这项工作将 在康奈尔大学兽医学院保拉·科恩博士的实验室进行，提供 在哺乳动物生殖生物学方面进行广泛的培训，特别是在减数分裂早期 配子发生。这一研究战略与申请者的培训和职业发展计划相结合，将 通过使用互补的模式生物，为减数分裂的研究提供了一种独特的方法。