Regulation of meiotic recombination and chromosome segregation in mammals

描述(由申请人提供)：在减数分裂过程中，DNA双链断裂修复和交叉形成是确保适当的染色体分离的关键。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体。然而，这些缺陷的分子基础还没有被很好地理解。这项提议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。我们确定了酵母CTF18基因的小鼠同源基因Chtf18在哺乳动物减数分裂中的新的重要作用。CTF18(染色体传递保真度因子18)编码一种进化上保守的蛋白质，它是复制因子C-样复合体CTF18-RLC的一部分。CTF18是酵母配子存活和染色体准确分离所必需的，也是果蝇生殖系发育和生育的关键。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力所需的称为粘附素的多蛋白复合体相互作用。然而，CTF18-RLC建立姐妹染色单体凝聚力的确切机制以及CHTF18在哺乳动物中的作用尚未阐明。以前，我们发现Chtf18在哺乳动物的雄性和雌性生殖系中都有表达，这表明Chtf18在配子发生中发挥了作用。最近，我们在体内证明了Chtf18在男性生育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期过早分离，DNA双链断裂的修复延迟，并持续到双胞体。此外，厚皮病的MLH1焦点(DNA交叉的标志)减少，提示交叉形成存在缺陷。最近，我们发现Chtf18缺失的雌性是不能生育的，在减数分裂I时分别过早地同源，并且Chtf18缺失的卵母细胞通过中期II的进程受到阻碍。因此，我们的数据显示了Chtf18在哺乳动物配子发生和减数分裂中的重要作用。我们推测，Chtf18通过与粘附素蛋白结合，促进DNA双链断裂修复和交叉形成，在减数分裂中发挥关键作用。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缺失小鼠过早同源分离的分子机制；(3)：研究Chtf18中断对染色体比对和分离的影响。我们提出的分子、细胞生物学和生化实验将为阐明Chtf18在哺乳动物减数分裂和减数分裂重组中的功能提供补充方法。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。

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.