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

基本信息

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
项目类别：
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

项目摘要

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.

概括染色体分离错误是人类非整倍性的主要原因，可能会带来灾难性的临床后果结果。减数分裂染色体分离的保真度需要精心策划的 DNA 双链破损修复。在此关键过程中，DNA 交叉和联会复合体将同源体束缚在一起染色体，姐妹染色单体的内聚力使姐妹染色单体保持在一起。然而，我们的差距还存在了解在同源染色体发生过程中建立和维持同源染色体束缚的机制减数分裂。我们的长期目标是定义调节正常染色体连锁和分离并识别导致染色体错误分离的分子缺陷。此前，我们确定了 CHTF18 作为哺乳动物减数分裂的重要调节因子。 CHTF18是一种保守的DNA复制必需蛋白对于酵母中的姐妹染色单体凝聚力和正确的染色体分离，雄性和雌性小鼠缺乏 Chtf18 (Chtf18-/-) 生育力低下，配子发生受损。在 Chtf18-/- 减数分裂细胞中，DNA 双链断裂（DSB）持续存在，DNA交叉减少，同源染色体过早分离。我们最近发现 CHTF18 是减数分裂染色体凝聚所必需的，这是一个介导的过程通过粘连蛋白。我们证明 CHTF18 与粘连蛋白发生物理相互作用并调节它们与染色质及其在前期 I 的组成。我们的数据支持 CHTF18 介导的模型减数分裂 DNA 复制和重组过程中的粘连蛋白促进交叉形成。然而， CHTF18 在整个减数分裂过程中的分布和时间尚不清楚，因为目前的抗体对局限性。 CHTF18 相互作用的功能伙伴也不清楚。这个基础信息是对于发现 CHTF18 在分子水平上如何发挥作用至关重要。因此，本提案的目标是生成并表征独特的小鼠品系，作为描述 CHTF18 在减数分裂中的作用的工具。这部小说小鼠品系将使我们能够克服阐明 CHTF18 作用机制的局限性缺乏特异性抗体。我们建议：1）生成并验证包含表位的小鼠品系 Chtf18 (Chtf18FH) 的标记等位基因并确定 CHTF18 在减数分裂中的时空分布；和 2) 识别并验证减数分裂中与 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，提供基础用于研究染色体凝聚力对于减数分裂重组和染色体的必要性的研究隔离。使用雄性和雌性小鼠将使我们能够精确定义雄性和雌性小鼠的性别二态性角色 CHTF18 在减数分裂的调控和时间安排中的作用。通过提供有关分布、时间的新颖信息，以及 CHTF18 在精母细胞和卵母细胞减数分裂关键步骤中的相互作用伙伴，这些研究将为定义 CHTF18 在这一关键发育过程中的分子机制提供了坚实的基础。这将使我们能够确定染色体错误分离和非整倍性的根本原因。