Development of a lacO/lacI based fluorescence reporter-operator system to study chromosome dynamics and double-strand break repair in mouse meiosis.

开发基于 lacO/lacI 的荧光报告操纵子系统，用于研究小鼠减数分裂中的染色体动力学和双链断裂修复。

• 批准号: 10674379
• 负责人: Roberto Jose Pezza
• 金额: $ 36.66万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-10 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10674379
• 关键词: 3-Dimensional; Affect; Aneuploidy; Biological Assay; Biological Process; Chromosome Pairing; Chromosome Segregation; Chromosomes; Co-Immunoprecipitations; Communities; Complement; Complex; Congenital Abnormality; Cytoskeleton; DNA Repair; Data; Development; Disease; Double Strand Break Repair; Down Syndrome; Dynein ATPase; Ensure; Event; Failure; Fertilization; Fluorescence; Gametogenesis; Genetic; Genetic Recombination; Genome engineering; Genomic Segment; Germ Cells; Goals; Homologous Gene; Human; Infertility; Karyotype; Kinesin; Knockout Mice; Knowledge; Lac Repressors; Location; MSH4 gene; Mammals; Measurement; Measures; Mediating; Meiosis; Meiotic Prophase I; Meiotic Recombination; Microscopy; Microtubules; Modeling; Molecular Motors; Motion; Motor; Movement; Mus; Nuclear Envelope; Physiological; Play; Pregnancy loss; Process; Productivity; Prophase; Proteins; Proteomics; Regulation; Reporter; Research; Resolution; Resources; Role; SPO11 gene; Seminiferous tubule structure; Sex Chromosomes; Site; Spermatocytes; Spontaneous abortion; Synaptonemal Complex; System; Testing; Time; Tissues; Translating; Visualization; autosome; cell behavior; chromosome missegregation; genome-wide; genomic locus; in silico; in vivo; inhibitor; movie; mutant; new technology; novel; pregnancy disorder; prevent; recruit; repaired; scaffold; segregation; simulation; telomere; tool

SUMMARY Fertilization of aneuploid gametes often leads to pregnancy loss or disorders such as Down’s syndrome. Aneuploidy occurs in 10-30% of human gametes and typically arises from chromosome segregation errors during meiosis. Meiotic events such as homologous chromosome pairing, double strand breaks (DSBs), recombination and the formation of crossovers, ensure proper chromosome segregation. Nevertheless, the mechanisms that regulate these events are incompletely understood. Consequently, there are currently few to no strategies to predict or prevent aneuploidy during gametogenesis. To overcome some limitations in the field, we established a fluorescence reporter-operator system (FROS) based on the lac operator-lac repressor (lacO-lacR) paradigm, which enables protein targeting to distinct genomic regions. Our long-term goal is to elucidate the mechanisms that regulate chromosome segregation during meiosis and thus ensure the formation of gametes with a normal karyotype. Aim 1 use FROS to dissect rapid chromosome motions in mouse spermatocytes at prophase I. We will also combine FROS with long-term 3D measurements of chromosome motions in seminiferous tubules to directly test whether homologous chromosome pairing arises from increasingly productive interactions (reeling in), versus reiterative rounds of transient interactions (catch and release). Additionally, we will analyze mutant spermatocytes by FROS to determine how key players, including components of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the synaptonemal complex, affect homologous and non-homologous interactions at specific loci. Dynein and microtubules interact with the LINC complex and are known to contribute to RPMs. Aim 2 will identify additional motors and cytoskeleton components that support these movements, building on our preliminary data uncovering kinesins as candidate molecular motors involved in generating RPMs. We will extend these findings with unbiased proteomic approaches, functional assays and FROS to decipher the dynamic forces that govern homolog pairing. Aim 3 will investigate the mechanism underlying the association between ANKRD31 and pro-DSB factors at recombination hotspots. Using FROS, we will target ANKRD31-GFP-lacI to the lacO region in spermatocytes, which we predict will recruit pro-DSB factors and recombination proteins. We will also analyze the effect of ANKRD31-GFP-lacI on downstream recombination intermediates at lacO sites. The proposed research will for the first time discern how chromosome context affects the mechanisms underlying chromosome segregation and thus advance new knowledge of fundamental meiotic processes and the causes of aneuploidy.

概括 非整倍体配子的受精通常会导致流产或唐氏综合症等疾病。 非整倍体发生在 10-30% 的人类配子中，通常是由染色体分离错误引起的 减数分裂事件，例如同源染色体配对、双链断裂 (DSB)、重组。 和交叉的形成，确保适当的染色体分离。 对这些事件的监管尚未完全了解，目前几乎没有策略。 为了克服该领域的一些限制，我们建立了预测或预防配子发生过程中的非整倍体。 基于 lac 操纵子-lac 阻遏物 (lacO-lacR) 范式的荧光报告操纵子系统 (FROS)， 这使得蛋白质能够靶向不同的基因组区域。我们的长期目标是阐明其机制。 在减数分裂过程中调节染色体分离，从而确保配子的形成具有正常的 目标 1 使用 FROS 剖析小鼠精母细胞在前期 I 的快速染色体运动。 还将 FROS 与生精小管中染色体运动的长期 3D 测量相结合， 直接测试同源染色体配对是否源于日益高效的相互作用（卷绕 in），与重复轮次的瞬时相互作用（捕获和释放）此外，我们将分析突变体。 通过 FROS 来确定精母细胞的关键参与者，包括核骨架连接器的组件 细胞骨架（LINC）复合体和联会复合体，影响同源和非同源 动力蛋白和微管与 LINC 复合物相互作用，并已知有贡献。 目标 2 将确定支持这些运动的其他电机和细胞骨架组件， 基于我们的初步数据，发现驱动蛋白作为参与生成的候选分子马达 我们将通过公正的蛋白质组学方法、功能测定和 FROS 将这些发现扩展到 破译控制同源配对的动力，目标 3 将研究其背后的机制。 我们将使用 FROS 来定位 ANKRD31 和 pro-DSB 因子在重组热点处的关联。 ANKRD31-GFP-lacI 到精母细胞中的 lacO 区域，我们预测它将招募 pro-DSB 因子并 我们还将分析 ANKRD31-GFP-lacI 对下游重组的影响。 拟议的研究将首次了解染色体背景如何影响。 染色体分离的机制，从而推进基本减数分裂的新知识 非整倍体的过程和原因。