Regulation of meiotic recombination and chromosome segregation in mammals

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是确保染色体正确分离的必要条件。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得到很好的理解。本研究的目的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。我们发现了酵母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（染色体传递保真因子18）编码一种进化上保守的蛋白，该蛋白是复制因子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殖的关键。在体外酵母和人类细胞中，CTF18-RLC与染色体在DNA复制过程中建立姐妹染色单体之间的内聚，并与染色体内聚所必需的多蛋白复合物（称为内聚蛋白）相互作用。然而，CTF18-RLC在哺乳动物中建立姐妹染色单体内聚的确切机制和CHTF18在哺乳动物中的作用尚未阐明。先前，我们发现Chtf18在哺乳动物雄性和雌性种系中都有表达，这表明Chtf18在配子发生中起作用。最近，我们在体内证明了Chtf18在雄性生殖和减数分裂中的作用。在chtf18缺失的小鼠中，减数分裂重组存在缺陷，同源染色体在前期i期过早分离。DNA双链断裂的修复被延迟，并持续到复合体。此外，在肺肿中，MLH1灶（DNA交叉标记）减少，表明交叉形成存在缺陷。最近，我们发现chtf18缺失的雌性不育，在减数分裂I中同源物过早分离，并且chtf18缺失的卵母细胞在中期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在减数分裂中发挥关键作用，通过与黏结蛋白结合促进DNA双链断裂修复和交叉形成。我们将通过三个实验目的来验证我们的假设：(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缺失小鼠早期同源性断裂的分子机制；(3)：研究Chtf18断裂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到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.