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.

项目摘要/摘要 调节基因表达何时何地，对于适当的发展，健康和生存力至关重要 在所有生物体中。调节基因表达的过程在广泛的范围内编排 来自分子尺度的空间和时间尺度，调节蛋白结合并解开DNA 在第秒至第二次尺度上，到核的组织，其中蛋白质和DNA形成动态 亚微米大小的域，这些域在几秒钟和几分钟内波动到这些事件的协调 在不同的组织类型中，数小时以及数百微米到毫米。尽管有动态 这些过程的性质，我们对它们的大多数知识都来自对固定样本的实验 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具有 授予量化基因调节动态的能力，并强调了 固定样品的研究。尽管这些新成像方法已经提供了出色的见解，但 由于技术限制，它们通常应用于在玻璃盖上生长的细胞，并与 它们已演变为功能的组织环境。 该提议的前提是，为了建立一个整体和定量的框架以了解基因 调节，我们必须开发和应用实验方法，以获取广泛的空间和 时间尺度涉及，并在内源性环境中这样做。为了实现这一目标，我建议将切割整合 边缘灯表显微镜，无标签的干涉法和分子成像工具，可以定量 单分子蛋白动力学，单个基因基因座的转录动力学，染色质动力学和 核在主动发展动物胚胎中的分室化。我将应用这些技术学习 果蝇Melanogaster胚胎早期发育过程中基因调节的动力学。这些胚胎 为研究基因调节的基本方面提供了理想的背景。他们从受精到 分化的组织仅在短短3个小时内逐渐逐渐 与整个胚胎的基因表达模式一起建立。我提出了利用的实验 我将开发出新的集成技术方法：（1）转录因子的动态如何 蛋白质 - 蛋白质和蛋白-DNA相互作用会影响其找到和结合其特定基因组靶标的能力 塑造核环境？ （2）如何在胚胎期间形成功能亚核室 发展，它们在塑造染色质动力学和基因表达模式中的作用是什么？ 这项建议将共同带来新的实验能力，这些功能将提供有关有关的基本见解 基因表达如何从分子尺度调节到生物尺度的动力学。这些新 集成数据集的类型将奠定基础，以开发一个定量和预测框架 可能使我们能够开发出新的治疗方法来纠正疾病中异常基因的表达。