Regulation of MutL-gamma Function in Mediating Crossing Over in Mammalian Meiosis

MutL-gamma 功能在哺乳动物减数分裂介导交换中的调节

• 批准号: 8759521
• 负责人: Paula Elaine Cohen
• 金额: $ 31.57万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8759521
• 关键词: 3-Dimensional; Accounting; Affect; Alleles; Antibodies; Binding; Cell Division Process; Chromosome Pairing; Chromosomes; Complex; Congenital Abnormality; Coupled; Cyclin-Dependent Kinases; Cyclins; DNA; DNA Binding; DNA biosynthesis; Diploidy; Dose; Double Strand Break Repair; Enhancers; Ensure; Event; Family member; Fertilization; Frequencies; Gene Transfer Techniques; Genes; Genetic Crossing Over; Genetic Recombination; Germ Cells; Grant; Haploidy; Heterozygote; Homologous Gene; Human; Image; Immunoprecipitation; MLH1 gene; MSH4 gene; Mammals; Mediating; Meiosis; Meiotic Prophase I; Mismatch Repair; Molecular; Mus; Mutant Strains Mice; Mutation; N-terminal; Normal Cell; Orthologous Gene; Pathway interactions; Phenocopy; Play; Ploidies; Preparation; Process; Proteins; Recruitment Activity; Regulation; Repair Complex; Reproduction; Resolution; Role; Site; Spermatocytes; Spontaneous abortion; Staging; Stem cells; Testing; Testis; Topoisomerase; Western Blotting; base; dosage; human CDK2 protein; innovation; mouse genome; mutant; novel; prenatal; programs; public health relevance; segregation

DESCRIPTION (provided by applicant): Crossing over during meiotic prophase I is essential for tethering homologous chromosomes together until the first meiotic division (MI). The localization of crossovers (CO) at the correct temporal and spatial frequency is critical for ensuring equal segregation of homologous chromosomes at MI, the importance of which is underscored by the observation that 50% of all spontaneous miscarriages in humans are due to chromosome mis-segregation errors at this stage. Not surprisingly, therefore, CO formation is tightly regulated, such that, of the 10-fold excess of initiating double strand break (DSB) events that arise during early prophase I, very few final COs are achieved. CO designation of DSB intermediates is largely controlled by the "ZMM" genes that include the meiosis-specific DNA mismatch repair heterodimeric complexes, MutSc (MSH4/MSH5) and MutLc (MLH1/MLH3). MutSc first binds to 150 of the 250+ DSBs and, of these, approximately 24-28 subsequently accumulate MutLc to become class I COs. How this specific subset of MutSc sites are designated is unclear, but recent studies in our lab have revealed that Cyclin N-terminal domain-containing protein-1 (CNTD1) plays a crucial role in this process, since MutSc focus frequency remains persistently elevated throughout prophase I in mouse mutants bearing a mutation in the Cntd1 gene. Moreover, MutLc fails to load on chromosomes during late prophase I in Cntd1 mutants, suggesting that CO designation and maturation are intimately coupled events regulated by CNTD1. Together with other regulators of crossover designation, including the Zip3/ZHP- 3 ortholog, RNF212, and human enhancer of invasion-10 (HEI10), we hypothesize that CNTD1 acts to ensure crossover designation in the class I CO pathway, either by promoting the processing/maturation of a finite set of MutSc events, or by removing excess DSB repair intermediates. Studies in this proposal are aimed at elucidating this novel crossover designation regulatory circuit. In aim 1, we will ask how and when CNTD1 is recruited to meiotic chromosome cores using a novel Cntd1-V5-tagged mouse, and by assessing CNTD1 localization in the presence of reduced levels of MutSc. In aim 2, we will identify key functional interactions that mediate CNTD1 function and we will determine whether CNTD1 acts in concert with one or several cyclin- dependent kinases, as its status as a cyclin family member would suggest. In aim 3, we will elucidate the mechanism by which loss of CNTD1 results in the absence of MutLg foci despite persistent MutSg complexes, asking whether the designation of COs in the mouse genome requires a dose-dependent threshold level of MutSc at specific DSB intermediate sites, whose accumulation and/or stability is ensured through the loading and activity of CNTD1. These studies take advantage of the impressive repertoire of mouse mutants available in the PI's lab, together with innovative experimental approaches that take advantage of advances in mouse transgenesis and high-resolution 3-dimensional imaging of prophase I events in mammalian germ cells.

描述（由申请人提供）：减数分裂前期 I 期间的交换对于将同源染色体束缚在一起直至第一次减数分裂 (MI) 至关重要。交叉 (CO) 在正确的时间和空间频率上的定位对于确保 MI 时同源染色体的平等分离至关重要，观察结果强调了这一点的重要性，即人类所有自发流产的 50% 是由于此阶段的染色体错误分离错误造成的。因此，毫不奇怪，CO 的形成受到严格调控，因此，在前期 I 早期出现的 10 倍过量的起始双链断裂 (DSB) 事件中，最终实现的 CO 很少。 DSB 中间体的 CO 命名主要由“ZMM”基因控制，其中包括减数分裂特异性 DNA 错配修复异二聚体复合物 MutSc (MSH4/MSH5) 和 MutLc (MLH1/MLH3)。 MutSc 首先与 250 多个 DSB 中的 150 个结合，其中大约 24-28 个随后积累 MutLc 成为 I 类 CO。 MutSc 位点的这个特定子集是如何指定的尚不清楚，但我们实验室最近的研究表明，细胞周期蛋白 N 末端结构域蛋白 1 (CNTD1) 在此过程中发挥着至关重要的作用，因为在携带 Cntd1 基因突变的小鼠突变体中，MutSc 聚焦频率在整个前期 I 中持续升高。此外，在 Cntd1 突变体的前期 I 晚期，MutLc 未能加载到染色体上，这表明 CO 指定和成熟是 CNTD1 调控的紧密耦合事件。与交叉指定的其他调节因子一起，包括 Zip3/ZHP-3 直系同源物、RNF212 和人类入侵增强子 10 (HEI10)，我们假设 CNTD1 通过促进有限组 MutSc 事件的处理/成熟，或通过去除多余的 DSB 修复中间体来确保 I 类 CO 途径中的交叉指定。该提案的研究旨在阐明这种新颖的交叉指定调节电路。在目标 1 中，我们将询问如何以及何时使用新型 Cntd1-V5 标记小鼠将 CNTD1 招募到减数分裂染色体核心，并在 MutSc 水平降低的情况下评估 CNTD1 定位。在目标 2 中，我们将确定介导 CNTD1 功能的关键功能相互作用，并将确定 CNTD1 是否与一种或多种细胞周期蛋白依赖性激酶协同作用，正如其作为细胞周期蛋白家族成员的地位所表明的那样。在目标 3 中，我们将阐明 CNTD1 丢失导致 MutLg 焦点缺失的机制，尽管 MutSg 复合物持续存在，询问小鼠基因组中 CO 的指定是否需要特定 DSB 中间位点处的剂量依赖性 MutSc 阈值水平，其积累和/或稳定性是通过 CNTD1 的负载和活性来确保的。这些研究利用了 PI 实验室中令人印象深刻的小鼠突变体库，以及利用小鼠转基因进展和哺乳动物生殖细胞前期 I 事件的高分辨率 3 维成像的创新实验方法。