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 作为这些决定的基础，即基因子集的转录激活，本质上是随机的。的 这项研究的目的是通过揭示动态基因是如何产生精确的模式来弥合这一差距。 activation.我们过去的工作主要集中在调控DNA序列的读出，如增强子， 控制靶基因的激活。然而，在上一个供资周期，我们确定了 这种调节发生的染色体距离很大，这一点很重要，常常导致 限制动态并增加随机性的额外来源。过去十年的许多研究表明， 他指出，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， 基因表达的调控。然而，染色质时间动力学的研究受到很大限制 由于使用固定材料的批量测定的普遍使用，并且传统的成像方法通常缺乏 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 通过开发新的成像方法和计算分析， 染色体结构图及其与转录的因果关系。为此，我们将 利用早期果蝇胚胎的优势，发展定量活体成像技术 方法.在这个系统中，分节基因表达的变化是细胞生长的位置特异性决定因素。 类型标识。因此，我们将研究苍蝇和哺乳动物特有的尺度上的调节相互作用（从 数十至数百种酶）及其在细胞特化背景下的意义， 有机体拟议的研究将有助于了解如何从细胞类型的规范出现强大的 随机基因表达，由长距离相互作用和染色质结构调节。以下 三个互补的假设进行了测试：1）不同的基因座特异性架构产生不同的功能 输出; 2）物理顺序是长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联系本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联系，这将最终引导我们调节和重新 工程师转录程序潜在的发展和疾病的过程。

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.