Positive and negative regulation of REC-8 cohesin during meiosis

减数分裂过程中REC-8粘连蛋白的正向和负向调节

• 批准号: 10292017
• 负责人: AARON F SEVERSON
• 金额: $ 44.55万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292017
• 关键词: Academic Research Enhancement Awards; Anaphase; Animals; Arabidopsis; Binding; Biological; C. elegans genome; CRISPR/Cas technology; Caenorhabditis elegans; Cell Nucleus; Cell Proliferation; Cells; Chromosome Segregation; Chromosomes; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Congenital Abnormality; Cytoplasm; Daughter; Defect; Ectopic Expression; Embryo; Ensure; Enzymes; Eukaryota; Excision; F Box Domain; Fertilization; Generations; Genes; Genetic; Genetic Screening; Genetic Transcription; Germ Cells; Haploidy; Human; Infertility; Inherited; Institution; Lead; Malignant Neoplasms; Mammals; Mediating; Mediator of activation protein; Meiosis; Methods; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Molecular Biology; Mutation; Oocytes; Organism; Pattern; Plants; Ploidies; Postdoctoral Fellow; Prognosis; Proliferating; Regulation; Reproductive Health; Research; Roberts-SC phocomelia syndrome; SKP Cullin F-Box Protein Ligases; Sister; Sister Chromatid; Spontaneous abortion; Students; Supervision; System; Transcriptional Regulation; Translational Regulation; Universities; Work; Yeasts; cohesin; cohesion; density; egg; experience; experimental study; genome editing; graduate student; inner city; innovation; man; morphogens; multidisciplinary; mutant; mutation screening; novel; novel strategies; prevent; protein complex; protein degradation; screening; segregation; separase; sperm cell; undergraduate student; underrepresented minority student; zygote

Project Summary/Abstract Precise reduction of chromosome copy number (the ploidy) from two to one is essential to create healthy sperm and eggs during meiosis. This requires two rounds of meiotic chromosome segregation. Homologous chromosomes separate first, sister chromatids second; this pattern requires two-step destruction of the α- kleisin subunit of the cohesin complexes that mediate sister chromatid cohesion (SCC). In most eukaryotes, replacing the mitotic α-kleisin Scc1/Rad21 with the meiosis-specific α-kleisin Rec8 is critical to establish the meiotic pattern of chromosome segregation (which reduces chromosome number) rather than the mitotic pattern (which stably maintains ploidy). Thus, specialized mechanisms must exist to ensure the establishment of REC-8-dependent SCC during meiosis and to prevent it during mitosis. The objectives of the proposed research are to identify the mediators of these antagonistic mechanisms and to understand their functions. Two specific aims will achieve these objectives. 1) We will identify the genes disrupted in five strains we isolated in an unbiased forward genetic screen for mutations that specifically disrupt REC-8- dependent SCC during C. elegans meiosis. We will then determine how the newly identified factors promote REC-8 cohesin function during normal meiosis. 2) We will determine whether PROM-1, a component of an SCF ubiquitin ligase known to target meiotic proteins for degradation, specifically marks nucleoplasmic, chromosomally unbound REC-8 cohesin for destruction prior to the onset of zygotic mitosis that begins following fertilization. We will also utilize CRISPR/Cas9-dependent genome editing to allow inducible REC-8 expression and determine the consequences of REC-8 expression in mitotically proliferating cells. The proposed research is innovative: although the importance of REC-8 cohesin for gamete formation has been established from yeast to man, the essential regulators that implement REC-8-dependent SCC during meiosis remain largely unidentified. Moreover, it is poorly understood in any organism how Rec8 cohesin function is prevented during mitotic proliferation: while known transcriptional and translational regulation prevent de novo REC-8 expression, additional mechanisms must prevent fertilized zygotes from inheriting REC-8 cohesin from the meiotic germline. We will apply novel approaches, many developed in my lab, to examine cohesin function and to edit the C. elegans genome. The research is significant: Defects in cohesin function are a major factor causing human miscarriage, birth defects, and infertility. Mutations in cohesin subunits and cohesin regulators are found in human cohesinopathies like Cornelia de Lange and Roberts-SC phocomelia syndromes. Cohesin misregulation and ectopic expression of meiosis-specific genes like REC-8 often correlates with poor prognosis in cancers. Thus, the relevance of the proposed research extends to human reproductive health and beyond. This multidisciplinary project will expose undergraduates to molecular biology, genetics, CRISPR-mediated genome editing, and applying the scientific method to understand complex biological problems.

项目摘要/摘要 精确地将染色体拷贝数(倍性)从两个减少到一个是创建健康的关键 减数分裂过程中的精子和卵子。这需要两轮减数分裂染色体分离。同源 染色体首先分离，姐妹染色单体第二；这种模式需要两步破坏α- 粘附素复合体的Kleisin亚基，介导姐妹染色单体凝聚力(SCC)。在大多数真核生物中， 用减数分裂特异的α-kleisin REC8取代有丝分裂的α-kleisin Scc1/Rad21是建立 染色体分离的减数分裂模式(减少染色体数目)，而不是有丝分裂 模式(稳定地保持倍性)。因此，必须存在专门的机制，以确保 REC-8依赖的SCC在减数分裂过程中的建立及其在有丝分裂过程中的预防。目标 建议的研究的目的是确定这些拮抗机制的介体并理解 它们的功能。有两个具体目标将实现这些目标。1)我们将在五年内确定被破坏的基因 我们在无偏正向遗传筛查中分离到的菌株，用于特异性破坏REC-8的突变- 线虫减数分裂过程中的体细胞依赖性。然后我们将确定新发现的因素是如何促进 Rec-8在正常减数分裂过程中发挥粘附素功能。2)我们将确定PROM-1是否为 已知的SCF泛素连接酶以减数分裂蛋白为目标进行降解，特别是标记核质， 在合子有丝分裂开始之前，染色体上未结合的REC-8粘附素被破坏 在受精之后。我们还将利用依赖CRISPR/Cas9的基因组编辑来允许可诱导的REC-8 并确定REC-8在有丝分裂增殖细胞中表达的后果。这个 提出的研究是创新的：尽管REC-8粘附素对配子形成的重要性一直是 从酵母到人，在减数分裂过程中实施依赖REC-8的SCC的基本调节因子 在很大程度上仍未确定。此外，在任何生物体中，对Rec8凝集素的功能都知之甚少 在有丝分裂增殖过程中被阻止：已知的转录和翻译调控阻止从头开始 REC-8表达，其他机制必须防止受精卵遗传REC-8粘附素 减数分裂胚系。我们将应用新的方法，其中许多是在我的实验室开发的，来检查凝集素功能 编辑线虫基因组。这项研究意义重大：凝集素功能缺陷是一个主要因素 导致人类流产、出生缺陷和不孕。粘附素亚基和粘附素调节因子的突变 在人类粘附素疾病中发现，如Cornelia de Lange和Roberts-SC Phocomelia综合征。粘合 REC-8等减数分裂特异基因的错误调控和异位表达通常与不良预后相关 在癌症方面。因此，拟议研究的相关性扩展到人类生殖健康和其他领域。 这个多学科的项目将使本科生接触到分子生物学、遗传学、CRISPR中介的 基因组编辑，并应用科学方法理解复杂的生物学问题。