Establishment and visualization of nuclear architecture at the onset of mammalian development

哺乳动物发育初期核结构的建立和可视化

• 批准号: 10264960
• 负责人: Maria Elena Torres Padilla
• 金额: $ 39.39万
• 依托单位: HELMHOLTZ ZENTRUM MUENCHEN - GMBH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-16 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10264960
• 关键词: 3-Dimensional; Address; Affect; Architecture; Biological Models; CRISPR imaging; Cell Cycle; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromatin Structure; Chromosomes; Communities; Competence; DNA; DNA Folding; Data; Dependence; Development; Developmental Process; Elements; Embryo; Epigenetic Process; Fertilization; Gene Expression; Gene Expression Regulation; Genetic Recombination; Genome; Genomic Instability; Genomic Segment; Genomic approach; Goals; Grant; Hereditary Disease; Image; KDM5B gene; Knowledge; Mammals; Mechanics; Methodology; Methods; Molecular; Mus; Nuclear; Nuclear Lamina; Nuclear Matrix; Organism; Pathway interactions; Physiological; Play; Pre-implantation Embryo Development; Process; Regulation; Role; Structural Protein; Technology; Time; Transcription Process; Validation; Visualization; base; biophysical properties; blastocyst; design; epigenome; epigenomics; imaging approach; imaging system; in vivo; in vivo imaging; innovation; insight; mammalian genome; novel; nuclear imaging; programs; scaffold; screening; structural genomics; tool; tool development; zygote

Project Summary/Abstract “Establishment and visualization of nuclear architecture at the onset of mammalian development” Nuclear organization is a key feature of our epigenome. The way in which our DNA is folded into the cell nucleus can affect gene expression and determine cellular identity, but it can also regulate recombination and genome instability. Indeed, the disruption of key elements underlying the architecture of the nucleus, such as Topologically-Associated Domain (TAD) boundaries, has been shown to be the cause of genetically inherited diseases in some instances. How nuclear organization is first established after fertilization in mammals is largely unknown. In particular, the lack of approaches to observe in real time the different layers of nuclear organization has been a barrier for the full understanding of the dynamics and the impact of nuclear organization in vivo. Most importantly, the chromatin and structural pathways that regulate the initial folding of our genome into the nucleus as well as the impact of the resulting genome organization for key developmental and transcriptional processes in vivo, is largely unknown. We have recently shown that nuclear organization, particularly the association of specific genomic regions to the nuclear lamina, is established de novo in the newly formed embryo immediately after fertilization. Lamina- Associated Domains (LADs) precede the consolidation of TADs and compartments, suggesting that the association with the nuclear lamina may be the first nuclear architectural scaffold and set the framework for nuclear architecture. Here, to fulfill the outstanding need for methodologies to visualize specific chromosome domains, we will establish “LivePaint”, a technology to visualize the fundamental pillars of genome organization and their dynamics in real time (Aim 1). We propose to image the process of establishment of nuclear architecture by applying this tool to the mouse pre-implantation embryo, a full organism amenable to imaging and in which real time dynamics can be studied in their natural context. In parallel, we will deploy low input genomic approaches, such as DamID to identify the regulators of LAD establishment (Aim 2). Finally, using this information, we will perform perturbations to establish the role of LADs in regulating the establishment of nuclear architecture through both imaging and genomics approaches (Aim 3). We will also identify the impact of the disruption to the establishment of nuclear organization in vivo on epigenetic features, such as replication timing and gene expression and ultimately their impact on developmental competence. Through the perturbation and live imaging of the main determinants of nuclear organization in vivo, this proposal will establish the functional dependencies between the different layers of nuclear organization and will reveal their role in cellular function, gene expression and developmental progression at the beginning of mammalian development. The knowledge generated through these Aims will address outstanding challenges for our understanding of how the mammalian genome folds within the nuclear space in a physiological context and will reveal molecular regulators of this process.

项目总结/摘要 “核结构的建立和可视化 在哺乳动物开始发育时” 核组织是我们表观基因组的一个关键特征。我们的DNA折叠进入细胞核的方式 可以影响基因表达并决定细胞身份，但它也可以调节重组和基因组 不稳定事实上，破坏核心结构的关键元素，如 拓扑相关结构域（Topologically Associated Domain，简称EAN）边界，已被证明是遗传性遗传缺陷的原因。 在某些情况下，疾病。 哺乳动物受精后细胞核组织最初是如何建立起来的，在很大程度上还不清楚。特别是 缺乏真实的实时观察核组织不同层次的方法， 充分了解体内细胞核组织的动力学和影响。最重要的是， 和结构途径，调节我们的基因组进入细胞核的初始折叠，以及 体内关键发育和转录过程的基因组组织主要是 未知 我们最近的研究表明，细胞核的组织，特别是特定基因组区域与细胞核的联系， 核纤层是受精后立即在新形成的胚胎中重新建立的。板层- 相关结构域（LAD）先于TAD和隔室的巩固，这表明 与核板层的结合可能是第一个核结构支架，并为核板层的形成提供了框架。 核结构 在这里，为了满足可视化特定染色体域的方法的突出需求，我们将 建立“LivePaint”，一种可视化基因组组织及其基本支柱的技术。 真实的动态（目标1）。我们建议通过以下方式来描绘核结构建立的过程： 将这种工具应用于小鼠植入前胚胎，一个完整的有机体适合成像，其中真实的 时间动力学可以在其自然环境中进行研究。与此同时，我们将部署低投入的基因组方法， 例如DamID，以识别法援署机构的规管机构（目的2）。利用这些信息，我们将 进行扰动，以确定LAD在调节核结构建立中的作用， 成像和基因组学方法（目标3）。我们还将确定中断对 核组织在体内的建立取决于表观遗传特征，例如复制时间和基因 表达，并最终影响发展能力。 通过对体内核组织主要决定因素的扰动和实时成像，该提议 将建立核组织不同层次之间的功能依赖关系，并将揭示 它们在哺乳动物早期细胞功能、基因表达和发育过程中的作用 发展 通过这些目标产生的知识将解决我们如何理解的突出挑战 哺乳动物基因组在生理环境中在核空间内折叠， 这个过程的监管者。