The Biophysical and Molecular Mechanisms of Reliability in Development

项目摘要 后生动物的发育依赖于精确和可重复的细胞命运决定。然而，分子事件 在这些决定的背后，即基因亚集的转录激活，天生是随机的。这个 这项拟议的研究的目标是通过揭示动态基因如何产生精确的模式来弥合这一差距 激活。我们过去的工作主要集中在读出调控DNA序列，如增强子， 控制目标基因的激活。然而，在前一个资金周期中，我们确定了功能 这一调控发生的大染色体距离的重要性，往往导致- 限制动力学，为随机性增加一个额外的来源。在过去的十年中，大量的研究表明 指出这种长距离染色体相互作用的普遍存在增加了另一层复杂性 基因表达的调节。然而，染色质的时间动力学研究是非常有限的。 由于普遍使用固定材料的批量分析，传统的成像方法往往缺乏 时空分辨率，以准确捕捉基因活动的动态。在这里，我们建议克服 这些限制通过开发新的成像方法和计算分析来提供动态 染色体结构图及其与转录的因果关系。为此，我们将 利用果蝇早期胚胎的优势进行定量活体成像 方法：研究方法。在这个系统中，片段基因表达的变化是细胞- 键入Identity。因此，我们将研究苍蝇和哺乳动物特有的尺度上的调控相互作用(来自 几十到几百个千碱基)以及它们在发展中的细胞规范的上下文中的含义 有机体。拟议的研究将有助于理解健壮的细胞类型规范是如何从 受远距离相互作用和染色质结构调控的随机基因表达。以下是 检验了三个互补性假设：1)不同的位点特定的结构产生不同的功能 输出；2)物理顺序是远距离染色体关系的基础；3)存在功能关系 染色质动态和活性之间的关系。这个项目的总体目标是建立一个量化的联系 染色质结构和转录活性之间的关系，这最终将导致我们调节和重新- 在发育和疾病过程中设计转录程序。

Project Summary Metazoan development relies on precise and reproducible cell fate decisions. However, the molecular events underlying these decisions, namely transcriptional activation of subsets of genes, are inherently stochastic. The goal of the proposed study is to bridge this gap by uncovering how precise patterns emerge from dynamic gene activation. Our past work was mostly focused on the readout of regulatory DNA sequences, such as enhancers, controlling the activation of a target gene. However, during the previous funding cycle, we identified the functional importance of the large chromosomal distances over which this regulation takes place, contributing to often rate- limiting dynamics and adding an extra source for stochasticity. Numerous studies in the past decade have pointed to the prevalence of such long-range chromosomal interactions adding another layer of complexity to the regulation of gene expression. However, investigation of temporal dynamics of chromatin is strongly limited by the prevalent use of bulk assays using fixed material, and traditional imaging methods often lack the spatiotemporal resolution to accurately capture the dynamics of gene activity. Here we propose to overcome these limitations by developing new imaging approaches and computational analyses to provide a dynamic picture of chromosomal architecture and its causal relationship to transcription. For this purpose, we will capitalize on the advantages of the early Drosophila embryo for the development of quantitative live imaging methods. In this system, changes in segmentation gene expression are position-specific determinants of cell- type identity. We will thus examine regulatory interactions at scales characteristic to flies and mammals (from tens to hundreds of kilobases) and their implications in the context of cellular specification in a developing organism. The proposed studies will help understand how robust cell type specification emerges from the stochastic gene expression that is regulated by long-range interactions and chromatin architecture. The following three complementary hypotheses are tested: 1) distinct locus-specific architectures generate different functional outputs; 2) physical order underlies long-distance chromosomal relationships; 3) a functional relationship exists between chromatin dynamics and activity. The overall goal of this project is to establish a quantitative link between chromatin architecture and transcriptional activity, which will ultimately lead us to regulate and re- engineer transcriptional programs underlying development and disease processes.