Generation and characterization of a novel mouse line to elucidate CHTF18 function in male and female meiosis

新型小鼠品系的产生和表征，以阐明 CHTF18 在雄性和雌性减数分裂中的功能

• 批准号: 10452959
• 负责人: KAREN MICHELE BERKOWITZ
• 金额: $ 7.41万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-16 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452959
• 关键词: Alleles; Aneuploidy; Antibodies; Antigens; Area; Assisted Reproductive Technology; Biochemical; C-terminal; CRISPR/Cas technology; Cells; Chromatin; Chromosome Cohesion; Chromosome Segregation; Chromosomes; Clinical; Couples; Cytology; DNA; DNA Double Strand Break; DNA biosynthesis; Data; Defect; Developmental Process; Double Strand Break Repair; Epitopes; Female; Foundations; Future; Gametogenesis; Generations; Genetic Recombination; Goals; Hemagglutinin; Histologic; Human; Impairment; Knowledge; Light; Mediating; Meiosis; Meiotic Prophase I; Meiotic Recombination; Methods; Microscopic; Mitosis; Modeling; Molecular; Multiprotein Complexes; Mus; Newborn Infant; Oocytes; Outcome; Pregnancy; Process; Proteins; Proteomics; Regulation; Research; Resolution; Role; Sister Chromatid; Specificity; Spermatocytes; Spontaneous abortion; Synaptonemal Complex; Testing; Yeasts; antibody detection; blastocyst; chromosome missegregation; cohesin; cohesion; experimental study; male; novel; premature; protein expression; protein function; sexual dimorphism; spatiotemporal; stillbirth; tool

SUMMARY Chromosome segregation errors are a leading cause of aneuploidy in humans that can have catastrophic clinical consequences. The fidelity of meiotic chromosome segregation requires well-orchestrated DNA double-strand break repair. During this critical process, DNA crossovers and the synaptonemal complex tether homologous chromosomes, and sister-chromatid cohesion keeps sister chromatids together. However, there is a gap in our knowledge of the mechanisms that establish and maintain the tethering of homologous chromosomes during meiosis. Our long-term goal is to define the mechanisms that regulate normal chromosome linkage and disjunction and identify molecular defects that cause chromosome mis-segregation. Previously, we identified CHTF18 as a crucial regulator of mammalian meiosis. CHTF18 is a conserved DNA replication protein essential for sister chromatid cohesion and proper chromosome segregation in yeast, Male and female mice lacking Chtf18 (Chtf18-/-) are subfertile with impaired gametogenesis. In Chtf18-/- meiotic cells, DNA double-strand breaks (DSBs) persist, DNA crossovers are decreased, and homologous chromosomes separate prematurely. We have recently discovered that CHTF18 is required for meiotic chromosome cohesion, a process mediated by cohesins. We showed that CHTF18 physically interacts with cohesins and regulates their association with chromatin and their composition during prophase I. Our data support a model in which CHTF18 mediates cohesins during meiotic DNA replication and recombination to promote crossover formation. However, the distribution and timing of CHTF18 throughout meiosis is not known because current antibodies pose a significant limitation. Functional partners with which CHTF18 interact are also not known. This foundational information is essential to discover how CHTF18 functions at the molecular level. Thus, the objective of this proposal is to generate and characterize a unique mouse line as a tool to delineate the role of CHTF18 in meiosis. This novel mouse line will permit us to overcome the limitation of elucidating the CHTF18 mechanism of action caused by a lack of specific antibodies. We propose to: 1) generate and validate a mouse line that contains an epitope- tagged allele of Chtf18 (Chtf18FH) and determine the spatiotemporal distribution of CHTF18 in meiosis; and 2) identify and validate functional partners that interact with CHTF18 in meiosis. Completing this project will generate an invaluable experimental tool and essential information about CHTF18 function, providing the basis for studies that investigate how chromosome cohesion is necessary for meiotic recombination and chromosome segregation. The use of male and female mice will allow us to precisely define the sexually dimorphic roles of CHTF18 in the regulation and timing of meiosis. By providing novel information about the distribution, timing, and interacting partners of CHTF18 during key steps of meiosis in spermatocytes and oocytes, these studies will provide a firm basis to define the molecular mechanisms of CHTF18 roles in this critical developmental process. This will allow us to establish underlying causes of chromosome mis-segregation and aneuploidy.

概括 染色体隔离错误是人类非整倍性的主要原因，可能患有灾难性的临床 结果。减数分裂染色体隔离的保真度需要精心策划的DNA双链 休息。在这个关键过程中，DNA跨界和突发性复合物系在一起 染色体和姐妹染色剂的凝聚力使姐妹染色质保持在一起。但是，我们的差距 了解建立和维持同源染色体的机制 减数分裂。我们的长期目标是定义调节正常染色体连锁和的机制 分离并确定导致染色体错误分离的分子缺陷。以前，我们确定了 CHTF18是哺乳动物减数分裂的关键调节剂。 CHTF18是一种保守的DNA复制蛋白必不可少的 对于酵母中的姐妹染色单体内聚力和适当的染色体隔离，雄性和雌性小鼠缺乏 CHTF18（CHTF18 - / - ）是属于配子发生受损的属。在CHTF18 - / - 减数分裂细胞中，DNA双链 断裂（DSB）持续存在，DNA交叉减少，同源染色体过早分开。 我们最近发现，减数分裂染色体内聚力需要CHTF18，这是介导的过程 通过粘着蛋白。我们表明CHTF18与粘着素进行物理相互作用，并调节它们与 染色质及其在预言I期间的组成。我们的数据支持CHTF18介导的模型 减数分裂DNA复制和重组期间的粘着蛋白，以促进交叉形成。但是， 在整个减数分裂中，CHTF18的分布和时机尚不清楚，因为电流抗体显着 局限性。与CHTF18相互作用的功能伙伴也不知道。这个基本信息是 发现CHTF18在分子水平上的功能至关重要。因此，该提议的目的是 生成并表征独特的鼠标线作为描述CHTF18在减数分裂中的作用的工具。这本小说 鼠标线将使我们能够克服阐明CHTF18作用机理的局限性 缺乏特定的抗体。我们建议：1）生成并验证包含表位的小鼠线 标记了CHTF18（CHTF18FH）的等位基因，并确定减数分裂中CHTF18的时空分布；和2） 识别并验证与减数分裂中与CHTF18相互作用的功能伙伴。完成这个项目将 生成有关CHTF18功能的宝贵实验工具和基本信息，提供基础 对于研究染色体凝聚的研究，对于减数分裂重组和染色体是必需的 隔离。雄性和雌性小鼠的使用将使我们能够精确地定义 CHTF18在减数分裂的调节和时机中。通过提供有关分布，时机的新颖信息， 以及在精子细胞和卵母细胞减数分裂的关键步骤中CHTF18的互动伴侣，这些研究将 提供牢固的基础来定义CHTF18在这个关键发展过程中角色的分子机制。 这将使我们能够建立染色体错误分离和非整倍的根本原因。