Quantifying the dynamics of gene regulation and nuclear organization during embryogenesis

量化胚胎发生过程中基因调控和核组织的动态

• 批准号: 10241709
• 负责人: Mustafa Aized Hasan Mir
• 金额: $ 158.4万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-21 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241709
• 关键词: Affect; Aging; Animals; Binding; Binding Proteins; Cell Nucleus; Cells; Chromatin; DNA; DNA-Protein Interaction; Data; Data Set; Defect; Development; Disease; Drosophila melanogaster; Embryo; Embryonic Development; Environment; Event; Fertilization; Foundations; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Glass; Goals; Grant; Health; Hour; Imaging Device; Imaging Techniques; Individual; Interferometry; Kinetics; Knowledge; Label; Lead; Life; Light; Malignant Neoplasms; Microscopy; Nature; Nuclear; Organism; Population; Process; Proteins; Resolution; Role; Sampling; Shapes; Techniques; Technology; Time; Tissue Differentiation; Tissues; Work; experimental study; genetic regulatory protein; genomic locus; imaging approach; insight; millimeter; molecular imaging; molecular scale; novel therapeutic intervention; prevent; repaired; scale up; single molecule; transcription factor; zygote

PROJECT SUMMARY/ABSTRACT Regulating when and where genes are expressed is essential to the proper development, health, and viability of all living organisms. The processes that regulate gene expression are choreographed across a broad range of spatial and temporal scales spanning from molecular scales where regulatory proteins bind and unbind DNA at sub-second to second time scales, to the organization of the nucleus where proteins and DNA form dynamic sub-micrometer sized domains that fluctuate over seconds and minutes, to the coordination of these events across distinct tissue types over hours and across hundreds of micrometers to millimeters. Despite the dynamic nature of these processes, most of our knowledge about them comes from experiments on fixed samples that provide population and time-averaged data. Recently, the advent of high-resolution live imaging techniques have granted the ability to quantify the dynamics of gene regulation and have highlighted what has been missed by studies in fixed samples. Although these new imaging approaches have already provided remarkable insights, due to technical constraints they are generally applied to cells grown on glass coverslips and isolated from the tissue contexts in which they have evolved to function. The premise of this proposal is that in order to build a holistic and quantitative framework to understand gene regulation, we must develop and apply experimental approaches that access the broad range of spatial and temporal scales involved, and do so in endogenous contexts. To achieve this goal I propose to integrate cutting edge light-sheet microscopy, label-free interferometry, and molecular imaging tools that will allow quantification of single-molecule protein kinetics, transcriptional dynamics at individual gene loci, chromatin dynamics, and the compartmentalization of nuclei in actively developing animal embryos. I will apply these technologies to study the dynamics of gene regulation during early development in Drosophila Melanogaster embryos. These embryos provide an ideal context for studying fundamental aspects of gene regulation. They proceed from fertilization to differentiated tissue in around just 3 hours during which chromatin and nuclear organization is progressively established along with patterns of gene expression across the embryo. I propose experiments that leverage the new integrated technological approaches I will develop to ask: (1) How do the dynamics of transcription factor protein-protein and protein-DNA interactions affect their ability to find and bind their specific genomic targets and shape the nuclear environment? and (2) How are functional sub-nuclear compartments formed during embryonic development, and what is their role in shaping chromatin dynamics and gene expression patterns? Together this proposal will lead to new experimental capabilities that will provide fundamental insights on the dynamics of how gene expression is regulated from the molecular scale up to the organismal scale. These new types of integrated datasets will lay the foundations for developing a quantitative and predictive framework which may allow us to develop new therapeutic approaches for correcting aberrant gene expression in disease.

项目概要/摘要 调节基因表达的时间和地点对于正常发育、健康和活力至关重要 所有生物体。调节基因表达的过程是在广泛的范围内精心设计的 空间和时间尺度跨越分子尺度，其中调节蛋白结合和解离 DNA 在亚秒到秒的时间尺度上，蛋白质和 DNA 形成动态的细胞核组织 亚微米大小的域在几秒和几分钟内波动，以协调这些事件 在数小时内跨越不同的组织类型，跨越数百微米到毫米。尽管动态 这些过程的本质，我们对它们的大部分了解都来自于对固定样本的实验 提供人口和时间平均数据。最近，高分辨率实时成像技术的出现 授予了量化基因调控动态的能力，并强调了所遗漏的内容 固定样本研究。尽管这些新的成像方法已经提供了非凡的见解， 由于技术限制，它们通常应用于在玻璃盖玻片上生长并与细胞隔离的细胞。 它们进化出功能的组织环境。 该提案的前提是为了建立一个全面、定量的框架来理解基因 监管，我们必须开发和应用实验方法来访问广泛的空间和 涉及时间尺度，并且是在内生背景下进行的。为了实现这个目标，我建议整合切割 边缘光片显微镜、无标记干涉测量和可进行定量的分子成像工具 单分子蛋白质动力学、单个基因位点的转录动力学、染色质动力学以及 活跃发育的动物胚胎中细胞核的区室化。我将应用这些技术来学习 果蝇胚胎早期发育过程中基因调控的动态。这些胚胎 为研究基因调控的基本方面提供了理想的背景。它们从受精到 组织在大约 3 小时内分化，在此期间染色质和核组织逐渐形成 与整个胚胎的基因表达模式一起建立。我建议利用以下实验 我将开发新的综合技术方法来问：（1）转录因子的动态如何 蛋白质-蛋白质和蛋白质-DNA 相互作用影响它们寻找和结合特定基因组靶标的能力， 塑造核环境？ (2) 胚胎期功能性亚核区室是如何形成的？ 它们在塑造染色质动态和基因表达模式方面发挥什么作用？ 该提案将共同带来新的实验能力，为以下方面提供基本见解： 从分子尺度到生物体尺度基因表达如何调控的动态。这些新 类型的综合数据集将为开发定量和预测框架奠定基础 可能使我们能够开发新的治疗方法来纠正疾病中的异常基因表达。