Quantifying the dynamics of gene regulation and nuclear organization during embryogenesis

项目摘要/摘要 控制基因表达的时间和地点对于正常的发育、健康和生存是必不可少的。 在所有活的有机体中。调控基因表达的过程是在广泛的范围内精心设计的 从调节蛋白结合和解锁DNA的分子尺度跨越的空间和时间尺度 在亚秒到秒的时间尺度上，到细胞核的组织，其中蛋白质和DNA形成动态 在几秒和几分钟内波动的亚微米大小的域，到这些事件的协调 在几个小时内跨越不同的组织类型，跨越数百微米到毫米。尽管有动态 这些过程的本质，我们对它们的大部分知识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 被授予量化基因调控动态的能力，并强调了 在固定样本中进行研究。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，它们通常应用于生长在玻璃盖片上的电池，并与 它们已经进化到可以发挥作用的组织环境。 这一建议的前提是，为了建立一个全面的、定量的了解基因的框架 在监管方面，我们必须开发和应用试验性方法，以获得广泛的空间和 涉及时间尺度，而且是在内生环境中这样做的。为了实现这一目标，我建议将切割 边缘光片显微镜、无标记干涉测量法和分子成像工具，将允许量化 单分子蛋白质动力学、个别基因位点的转录动力学、染色质动力学和 动物胚胎活跃发育过程中细胞核的区域化。我将把这些技术应用于研究 果蝇胚胎早期发育过程中的基因调控动态。这些胚胎 为研究基因调控的基本方面提供了一个理想的背景。他们从受精到 在大约3小时内分化的组织，在此期间染色质和核组织逐渐 与整个胚胎的基因表达模式一起建立的。我建议进行一些实验，利用 我将开发新的集成技术方法来问：(1)转录因子的动力学是如何 蛋白质-蛋白质和蛋白质-DNA的相互作用影响它们寻找和结合其特定基因组靶点和 塑造核环境？以及(2)胚胎期间功能性亚核区是如何形成的 发展，以及它们在塑造染色质动力学和基因表达模式中扮演什么角色？ 总而言之，这项提议将带来新的实验能力，将为 从分子尺度到生物体尺度的基因表达调控的动力学。这些新的 各种类型的综合数据集将为开发量化和预测框架奠定基础，该框架 可能会让我们开发新的治疗方法来纠正疾病中异常的基因表达。

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.