Regulation of meiotic recombination and chromosome segregation in mammals

哺乳动物减数分裂重组和染色体分离的调控

• 批准号: 9118223
• 负责人: KAREN MICHELE BERKOWITZ
• 金额: $ 28.86万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-05 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9118223
• 关键词: Acetylation; Anaphase; Aneuploidy; Biochemical; Biological; CSPG6 gene; Cells; Chromosome Cohesion; Chromosome Segregation; Chromosomes; Cleaved cell; Co-Immunoprecipitations; Complex; Congenital Abnormality; DNA Double Strand Break; DNA Markers; DNA biosynthesis; Data; Defect; Development; Diplotene Stage; Double Strand Break Repair; Ensure; Enzymes; Etiology; Female; Fertility; Frequencies; Gametogenesis; Genes; Genetic; Genetic Recombination; Germ Cells; Glean; Goals; Health; Homologous Gene; Hot Spot; Human; Immunoprecipitation; In Vitro; Infertility; Knockout Mice; Lead; Light; MLH1 gene; Mammals; Mediating; Meiosis; Meiotic Prophase I; Meiotic Recombination; Metaphase; Mitosis; Molecular; Mus; Null Lymphocytes; Oocytes; Optic Chiasm; Pachytene Stage; Phenotype; Play; Pregnancy loss; Process; Proteins; Regulation; Role; Sister Chromatid; Spermatocytes; Staging; Testing; Yeasts; activator 1 protein; base; cohesin; cohesion; in vivo; male; premature; protein complex; repaired; research study; segregation; separase; transmission process; zygote

DESCRIPTION (provided by applicant): DNA double strand break repair and crossover formation during meiosis are essential to ensure proper chromosome segregation. Errors in these processes can lead to chromosome mis-segregation and ultimately, aneuploidy in humans. Yet the molecular basis of the defects is not well understood. The goal of this proposal is to shed new light on the defects occurring during meiotic recombination and chromosome segregation in mammals. We identified new and important roles for Chtf18, the murine orthologue of the yeast CTF18 gene, in mammalian meiosis. CTF18 (Chromosome Transmission Fidelity factor 18) encodes an evolutionarily conserved protein that is part of the replication factor C-like complex, CTF18-RLC. CTF18 is necessary for gamete viability and accurate chromosome segregation in yeast, and it is crucial for germline development and fertility in the fruitfly. CTF18-RLC associates with chromosomes to establish cohesion between sister chromatids during DNA replication, and interacts with multi-protein complexes necessary for chromosome cohesion, called cohesins, in both yeast and in human cells in vitro. However, the exact mechanism by which CTF18-RLC establishes sister chromatid cohesion and the role CHTF18 plays in mammals have not been elucidated. Previously, we showed that Chtf18 is expressed throughout the mammalian male and female germline, suggesting a role for Chtf18 in gametogenesis. Recently, we demonstrated a role for Chtf18 in male fertility and meiosis in vivo. In Chtf18-null mice, meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double strand breaks is delayed, and these persist into diplonema. In addition, MLH1 foci (markers of DNA crossovers) are decreased in pachynema, suggesting a defect in crossover formation. Recently, we found that Chtf18-null females are subfertile, homologues separately prematurely during meiosis I, and progression of Chtf18-null oocytes through metaphase II is impaired. Thus, our data demonstrate essential roles for Chtf18 in mammalian gametogenesis and meiosis. We hypothesize that Chtf18 plays a critical role in meiosis by facilitating DNA double strand break repair and crossover formation through association with cohesin proteins. We will test our hypothesis in three experimental aims: (1): to explore the role of Chtf18 in DNA double strand break repair and crossover formation; (2): to investigate the molecular mechanisms underlying premature homologue disjunction in Chtf18-null mice; (3): to examine the consequences of Chtf18 disruption on chromosome alignment and segregation. Our proposed molecular, cell biological, and biochemical experiments will provide complementary approaches to elucidate the functions of Chtf18 in mammalian meiosis and meiotic recombination. Information gleaned from our studies will ultimately elucidate the molecular etiologies underlying chromosome mis- segregation and aneuploidy in humans.

描述（由申请人提供）：减数分裂期间DNA双链断裂修复和交换形成对于确保正确的染色体分离至关重要。这些过程中的错误可能导致染色体错误分离，最终导致人类的非整倍性。然而，这些缺陷的分子基础还没有得到很好的理解。该建议的目标是揭示哺乳动物减数分裂重组和染色体分离过程中发生的缺陷。 我们确定了新的和重要的作用，Chtf 18，小鼠直系同源的酵母CTF 18基因，在哺乳动物减数分裂。CTF 18（染色体传递保真度因子18）编码一种进化上保守的蛋白质，是复制因子C样复合物CTF 18-RLC的一部分。CTF 18是酵母中配子存活和准确染色体分离所必需的，并且对于果蝇的生殖系发育和生育力至关重要。CTF 18-RLC在DNA复制过程中与染色体结合以建立姐妹染色单体之间的凝聚力，并在酵母和体外人类细胞中与染色体凝聚力所必需的多蛋白复合物（称为凝聚素）相互作用。然而，CTF 18-RLC建立姐妹染色单体凝聚力的确切机制以及CHTF 18在哺乳动物中的作用尚未阐明。以前，我们表明，Chtf 18在整个哺乳动物雄性和雌性生殖系中表达，这表明Chtf 18在配子发生中的作用。最近，我们证明了Chtf 18在体内雄性育性和减数分裂中的作用。在Chtf 18基因敲除小鼠中，减数分裂重组有缺陷，同源染色体在前期I过早分离。DNA双链断裂的修复被延迟，并持续到双丝体。此外，MLH 1病灶（DNA交换的标志物）减少粗线，表明在交叉形成的缺陷。最近，我们发现，Chtf 18-null女性不育，同源物分别过早减数分裂I期间，通过中期II的Chtf 18-null卵母细胞的进展受损。因此，我们的数据表明，在哺乳动物配子发生和减数分裂的重要作用的ChTF 18。我们推测，Chtf 18通过促进DNA双链断裂修复和交叉形成，通过与cohesin蛋白的协会在减数分裂中起着关键作用。我们将在三个实验目标中验证我们的假设：（1）：探索Chtf 18在DNA双链断裂修复和交叉形成中的作用;（2）：研究Chtf 18敲除小鼠中过早同源分离的分子机制;（3）：检查Chtf 18破坏对染色体排列和分离的影响。我们提出的分子，细胞生物学和生物化学实验将提供互补的方法来阐明哺乳动物减数分裂和减数分裂重组中的Chtf 18的功能。从我们的研究中收集的信息将最终阐明人类染色体错误分离和非整倍体的分子病因。