Mechanisms Driving Meiotic Chromosome Morphogenesis

减数分裂染色体形态发生的驱动机制

• 批准号: 10226866
• 负责人: Carolyn Rose Milano
• 金额: $ 6.64万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226866
• 关键词: ATP phosphohydrolase; Alleles; Automobile Driving; Binding; Binding Proteins; Binding Sites; Biology; Biotinylation; C-terminal; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromatin Loop; Chromosomal Instability; Chromosome Pairing; Chromosomes; Computational Biology; Congenital Abnormality; Cytology; DNA; DNA Repair; Deposition; Dissociation; Down Syndrome; Ectopic Expression; Edward' s syndrome; Elements; Environment; Excision; Exhibits; Fertility; Gene Expression; Genes; Genetic Recombination; Goals; Histones; Human; Incidence; Infertility; Investigation; Kinetics; Knowledge; Lateral; Length; Location; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediating; Meiosis; Meiotic Recombination; Methods; Mitotic; Modeling; Molecular; Morphogenesis; Mus; Mutation; Organism; Pathway interactions; Pattern; Positioning Attribute; Process; Proteins; Proteomics; Reading; Research; Research Personnel; Role; Saccharomyces cerevisiae; Saccharomycetales; Series; Signal Transduction; Site; Southern Blotting; Structure; Synapses; Synaptonemal Complex; Techniques; Testing; Time; Training; Universities; Work; Yeasts; base; cohesin; deletion library; egg; experience; experimental study; genome sequencing; human disease; insight; mutant; novel; prevent; programs; protein complex; recruit; repaired; scaffold; sperm cell; whole genome

Project Summary The overall goal of this proposal is to facilitate my aspiration to become a primary investigator of chromosome biology at a research-intensive university. This project will build on my graduate background in mammalian meiosis while expanding my technical abilities to facilitate my investigation of chromosome biology in other contexts, such as human disease. The proposed research utilizes S. cerevisiae to investigate the chromosome dynamics that occur prior to the first meiotic division. During this time, the nucleus is reorganized to allow for the alignment of homologous chromosomes. The proper alignment of homologous chromosomes depends on a series of molecular pathways, including endogenous DNA damage and repair, checkpoint signaling, and the construction of a structural scaffold known as the synaptonemal complex (SC). The SC is made of two lateral elements that form along the lengths of each chromosome and one central element that connect the lateral elements of homologous chromosomes. The conserved axis proteins Hop1 (HORMAD1/2 proteins in mice and humans) are an essential component of the lateral element and a central regulator of SC assembly. Despite extensive research, how the axis proteins localize to chromatin and then recruit the central element to build the SC remains unclear. Indeed, a large fraction of Hop1 dissociate from the lateral elements as the central element is deposited, raising the question whether Hop1 is a structural component of the SC or only needed to mediate deposition of the central element. Aim 1 utilizes conditional protein induction and depletion experiments to test the requirements of Hop1 in SC formation. It will also determine the role of Hop1 dissociation in this process. Aim 2 investigates the interface between DNA and the lateral element by using a novel hop1 separation-of-function allele that exhibits normal DSB induction and repair kinetics but altered DNA breakage patterns, which likely reflect altered Hop1 chromatin association. This allele of HOP1 removes an uncharacterized region in the center of Hop1 that shares features with PHD domains, which are canonically important for reading and responding to histone marks. Aim 2-1 uses this hop1 mutant to determine the effects the axis patterning by ChIP-seq of axis proteins and to correlate these effects with altered break patterning observed by whole-chromosome Southern blot analysis. Aim 2-2 utilizes targeted biotinylation and mass spectrometry to define the chromatin environment surrounding Hop1 binding sites. This experiment will be used to identify candidate proteins that will be tested for a role in axis protein positioning using the yeast deletion library. The results of this proposal will provide important new insight into the mechanism of meiotic chromosome morphogenesis and will serve as a framework for understanding infertility and birth defects in humans. This proposal has a strong training component and will expand my knowledge to whole genome sequencing techniques, computational biology, and proteomics. Altogether, this training will provide me with the experience needed for establishing an independent research program in chromosome biology.

项目摘要 该提议的总体目标是促进我成为染色体的主要研究者的愿望 研究密集于大学的生物学。这个项目将以我在哺乳动物的研究生背景为基础 减数分裂的同时扩展了我的技术能力，以促进我对其他染色体生物学的调查 情境，例如人类疾病。拟议的研究利用酿酒酵母研究染色体 在第一个减数分裂部门之前发生的动态。在此期间，核被重组以允许 同源染色体的对齐。同源染色体的适当比对取决于 一系列分子途径，包括内源性DNA损伤和修复，检查点信号传导以及 结构支架的构造称为突发事件复合物（SC）。 SC由两个侧面制成 沿每个染色体的长度形成的元素和连接侧面的一个中心元素 同源染色体的元素。保守的轴蛋白Hop1（小鼠和小鼠和 人类）是侧向元素的重要组成部分，也是SC组装的中心调节器。 尽管进行了广泛的研究，但轴蛋白如何定位到染色质，然后募集中央 构建SC的要素尚不清楚。实际上，很大一部分Hop1与横向元素分离 随着中心元素的沉积，提出了一个问题，hop1是SC的结构成分还是 仅需要调解中心元素的沉积。 AIM 1利用条件蛋白诱导和 耗尽实验以测试SC形成中Hop1的要求。它还将决定Hop1的作用 在此过程中解离。 AIM 2通过使用A 新型Hop1分离功能等位基因表现出正常的DSB诱导和修复动力学，但DNA改变了 破裂模式可能反映了Hop1染色质关联的改变。 Hop1等位基因删除了 Hop1中心的未表征区域，该区域与PhD域共享特征，它们在规范上是 对于阅读和响应组蛋白标记很重要。 AIM 2-1使用此Hop1突变体确定效果 轴蛋白的芯片序列形式的轴，并将这些效果与破裂图案的改变相关 通过全染色体Southern印迹分析观察到。 AIM 2-2利用靶向的生物素化和质量 光谱法定义了围绕Hop1结合位点的染色质环境。这个实验将是 用于鉴定候选蛋白，该蛋白质将在使用酵母中测试在轴蛋白定位中的作用 删除库。该提案的结果将为减数分裂机制提供重要的新见解 染色体形态发生，将作为理解不育和出生缺陷的框架 人类。该提议具有强大的培训组成部分，并将我的知识扩展到整个基因组 测序技术，计算生物学和蛋白质组学。总之，这项培训将为我提供 建立染色体生物学独立研究计划所需的经验。