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

基本信息

批准号：
8710284
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
项目类别：
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/8710284
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 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 public health relevance repaired research study segregation separase transmission process zygote

项目摘要

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）：减数分裂过程中 DNA 双链断裂修复和交叉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，并最终导致人类非整倍体。然而，这些缺陷的分子基础尚不清楚。该提案的目的是为哺乳动物减数分裂重组和染色体分离过程中发生的缺陷提供新的线索。我们确定了 Chtf18（酵母 CTF18 基因的小鼠直系同源物）在哺乳动物减数分裂中的新的重要作用。 CTF18（染色体传输保真因子 18）编码一种进化上保守的蛋白质，该蛋白质是复制因子 C 样复合物 CTF18-RLC 的一部分。 CTF18 对于酵母中的配子活力和准确的染色体分离是必需的，并且对于果蝇的种系发育和生育能力至关重要。 CTF18-RLC 与染色体结合，在 DNA 复制过程中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所需的多蛋白复合物（称为黏连蛋白）相互作用。然而，CTF18-RLC建立姐妹染色单体凝聚力的确切机制以及CHTF18在哺乳动物中发挥的作用尚未阐明。此前，我们发现 Chtf18 在整个哺乳动物雄性和雌性种系中表达，表明 Chtf18 在配子发生中发挥作用。最近，我们证明了 Chtf18 在体内男性生育力和减数分裂中的作用。在 Chtf18 缺失的小鼠中，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，并且这些断裂持续形成双线体。此外，粗线瘤中的 MLH1 焦点（DNA 交叉标记）减少，表明交叉形成存在缺陷。最近，我们发现 Chtf18 缺失的雌性生育力低下，同源体在减数分裂 I 期间过早分离，并且 Chtf18 缺失的卵母细胞通过中期 II 的进展受到损害。因此，我们的数据证明了 Chtf18 在哺乳动物配子发生和减数分裂中的重要作用。我们假设 Chtf18 通过与粘连蛋白结合促进 DNA 双链断裂修复和交叉形成，从而在减数分裂中发挥关键作用。我们将从三个实验目标来检验我们的假设：（1）：探索Chtf18在DNA双链断裂修复和交叉形成中的作用； (2)：研究Chtf18缺失小鼠同源体过早分离的分子机制； (3)：检查 Chtf18 破坏对染色体排列和分离的影响。我们提出的分子、细胞生物学和生化实验将提供补充方法来阐明 Chtf18 在哺乳动物减数分裂和减数分裂重组中的功能。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性的分子病因。