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: 10313341
• 负责人: Ian Wolff
• 金额: $ 6.6万
• 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/10313341
• 关键词: Investigating; conserved; mechanisms; orchestrate; prophase

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.

有性生殖生物通过减数分裂产生配子，减数分裂是遗传物质产生的过程。 细胞减半形成单倍体精子或卵子，尽管减数分裂对于所有动物生命都是至关重要的。 容易出错，估计所有人类受孕的 5-25% 会产生非整倍体胚胎，通常会导致 严重的发育缺陷和妊娠并发症每年影响数百万人。 因此，研究定义减数分裂进展的核心分子机制至关重要。 进一步了解可能存在哪些检查点来感知错误。 使用两种强大的减数分裂模型（实验室小鼠小家鼠和线虫）进行减数分裂进展 秀丽隐杆线虫减数分裂 I 通过同源物的配对、联会和交叉 (CO) 进行。 染色体在前期，只有当这些事件正确发生后，同源物才能对齐 适当地发生在减数分裂纺锤体上，然后在第一次减数分裂时均匀分离。然而，尚不清楚。 细胞周期机制如何成功感知 CO 形成以将减数分裂推进到适当的 CO 阶段。 形成需要小鼠精母细胞中含有蛋白质 CNTD1 的直系同源细胞周期蛋白样结构域，并且 COSA-1，在线虫卵子发生过程中，CNTD1 与泛素 E2 结合物 CDC34 相互作用。 与 E3 泛素连接酶的 Skp1-Cullin-F-Box (SCF) 家族一起作用的酶，是 CDC34 的已知靶标。 有丝分裂系统是细胞周期 M 期抑制剂，WEE1 在 CNTD1 敲除小鼠精母细胞中，CDC34 是。 细胞核中不存在，并且 WEE1 异常地持续存在直至减数分裂前期结束，这表明其具有一定的作用 CNTD1 促进 WEE1 降解中的交叉成熟并导致进入 M 期。 导致这样的假设：从减数分裂前期 I 到 M 期 I 的进展是通过直接许可获得的 SCFCDC34 以交叉依赖性方式泛素化 WEE1，这种方式在真核生物中是保守的。 该提案提出了两个具体目标：1) SCFCDC34 介导的 WEE1 泛素化对于进展至关重要吗？ 从减数分裂前期 I 到 M 期 I？ 将开发一种直接在野生型和交叉缺陷突变小鼠中进行测试的方法。 可视化线虫中 WEE1 直向同源物的种系丰度，WEE-1.3，以测试 WEE-1.3 在 线虫减数分裂进程 2) SCFCDC34 介导的 WEE1 的生化机制是什么。 SCFCDC34复合物的分子成分将在小鼠精母细胞中被阐明 通过对假定的复杂成员的突变分析，以及通过 RNAi 筛选秀丽隐杆线虫，这项工作将。 在康奈尔大学兽医学院 Paula Cohen 博士的实验室中进行，提供 哺乳动物生殖生物学的广泛培训，特别是早期减数分裂事件 该研究策略与申请人的培训和职业发展计划相结合。 通过使用互补模型生物体提供了研究减数分裂的独特方法。