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: 10574577
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10574577
• 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 Double Strand Break; DNA biosynthesis; Data; Defect; Developmental Process; Double Strand Break Repair; Epitopes; Female; Future; Gametogenesis; Generations; Genetic Crossing Over; Genetic Recombination; Goals; Hemagglutinin; Histologic; Human; Impairment; Knowledge; 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; 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; ultra high resolution

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.

总结 染色体分离错误是人类非整倍体的主要原因，其可以具有灾难性的临床表现。 后果减数分裂染色体分离的精确性需要精心安排的DNA双链 破损修复在这一关键过程中，DNA交换和联会复合体同源系链 姐妹染色单体凝聚力使姐妹染色单体保持在一起。然而，我们的差距是， 建立和维持同源染色体系留的机制的知识， 减数分裂我们的长期目标是确定调节正常染色体连锁的机制， 分离和鉴定导致染色体错误分离的分子缺陷。此前，我们发现 CHTF 18作为哺乳动物减数分裂的重要调节因子。CHTF 18是一个保守的DNA复制蛋白， 对于酵母中的姐妹染色单体凝聚和正确的染色体分离，雄性和雌性小鼠缺乏 Chtf 18（Chtf 18-/-）是低生育力的，配子发生受损。在Chtf 18-/-减数分裂细胞中，DNA双链 断裂（DSB）持续存在，DNA交换减少，同源染色体过早分离。 我们最近发现，CHTF 18是减数分裂染色体凝聚所必需的， cohesins的。我们发现，CHTF 18与粘附素发生物理相互作用，并调节它们与蛋白质的结合。 染色质及其组成在前期I。我们的数据支持CHTF 18介导的模型 在减数分裂DNA复制和重组过程中的粘着蛋白，以促进交换形成。但 CHTF 18在整个减数分裂中的分布和时间是未知的，因为目前的抗体对CHTF 18的表达具有显著的影响。 限制.与CHTF 18相互作用的功能性伴侣也是未知的。这些基本信息是 这对于发现CHTF 18在分子水平上的功能至关重要。因此，本提案的目的是 产生并表征独特的小鼠系作为描述CHTF 18在减数分裂中的作用的工具。这本小说 小鼠系将允许我们克服阐明CHTF 18作用机制的局限性， 缺乏特异性抗体。我们提出：1）产生并验证含有表位的小鼠系- 标记Chtf 18的等位基因（Chtf 18FH），并确定CHTF 18在减数分裂中的时空分布;和2） 鉴定和验证在减数分裂中与CHTF 18相互作用的功能伴侣。完成该项目将 生成一个宝贵的实验工具和关于CHTF 18功能的基本信息，提供基础 对于研究染色体凝聚力如何对减数分裂重组和染色体 隔离。雄性和雌性小鼠的使用将使我们能够精确地定义 CHTF 18在减数分裂的调节和定时中的作用。通过提供关于分布，时间， 以及在精母细胞和卵母细胞减数分裂的关键步骤中CHTF 18的相互作用伴侣，这些研究将 为确定CHTF 18在这一关键发育过程中作用的分子机制提供了坚实的基础。 这将使我们能够建立染色体错误分离和非整倍体的根本原因。