The Biophysical and Molecular Mechanisms of Reliability in Development

 描述(由申请人提供)：细胞命运决定导致后生动物发育的高度可重复性的模式。然而，这些决定取决于系统在分子水平上克服随机转录事件。这项拟议的研究的目标是建立活的苍蝇胚胎转录的分子机制，并了解这些机制的动态和随机性质，最终导致精确的模式。为了深入了解跨越如此巨大的不同时间和空间尺度的调控过程-分子转录事件与宏观模式的形成-我们需要在DNA调控元件和细胞命运决定之间建立联系，在稳定的转录输出方面，在时间和空间上都能分辨。因此，我们将使用定量测量来确定调控DNA元件和转录活性之间的关系，以定义一组表征转录动力学的参数，例如聚合酶负载量和延伸率。这种关系将为我们提供序列和命运之间的联系，并将使我们能够建立量化模型，从而得出可在体内直接检验的预测。我们提出了三种方法，有助于在不同和互补的水平上建立这些结构-功能关系：1)我们将剖析小调控增强子的结构，并测量报告蝇构建体中的转录活性和输出。2)利用基因组编辑，我们将测量针对特定基因的增强子和增强子组合的内源转录活性。3)我们将把这种方法扩大到固定组织中的大量基因，以根据基因的动态转录特性识别基因类别。建立定量的结构-功能关系最终将导致我们规范和 重新设计作为发育和疾病过程基础的转录程序。

 DESCRIPTION (provided by applicant): Cell fate decisions lead to highly reproducible patterns in metazoan development. However, these decisions hinge upon the system overcoming stochastic transcriptional events at the molecular level. The goal of the proposed study is to establish the molecular mechanisms underlying transcription in living fly embryos and to understand the dynamic and stochastic nature of these mechanisms that eventually lead to precise patterns. To gain insights into the regulatory processes that span such vastly differing temporal and spatial scales-molecular transcription events vs. formation of macroscopic patterns-we need to establish a connection between DNA regulatory elements and cell fate decisions in terms of stable transcriptional output, resolved in both time and space. Therefore we will determine a relationship between regulatory DNA elements and transcription activity using quantitative measurements to define a set of parameters characterizing transcription dynamics, such as, e.g., polymerase loading and elongation rates. Such a relationship will give us a link between sequence and fate, and will allow us to formulate quantitative models that lead to predictions that are directly testable in vivo. We propose three approaches that will contribute to the establishment of these structure- function relationships at different and complementary levels: 1) we will dissect the structure of small regulatory enhancers and measure the transcription activity and output in reporter fly constructs. 2) Using genome editing we will measure the endogenous transcription activity of enhancers and combinations of enhancers for specific genes. 3) We will scale up the approach to large numbers of genes in fixed tissue to identify classes of genes according to their dynamic transcription properties. Establishing a quantitative structure-function relationship will ultimately lead us to regulate and re-engineer the transcription programs underlying development and disease processes.