Uncovering the fundamental principles of transcriptional regulation

揭示转录调控的基本原理

• 批准号: 9751337
• 负责人: Robert Charles Brewster
• 金额: $ 41.88万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9751337
• 关键词: Animal Model; Binding; Binding Sites; Cells; Code; Complex; DNA; Decision Making; Disease; Dissection; Escherichia coli; Feedback; Foundations; Gene Expression; Gene Expression Regulation; Genes; Genome; Individual; Investigation; Location; Measures; Methods; Nucleic Acid Regulatory Sequences; Pattern; Play; Process; Proteins; Regulation; Regulatory Element; Role; Scheme; Specific qualifier value; Stimulus; Theoretical model; Time; Transcriptional Regulation; Work; base; biophysical model; design; experimental study; fitness; genomic data; recruit; response; synthetic biology; transcription factor

Project summary/Abstract The regulatory code, inscribed in DNA, specifies how the expression of a gene will be tuned by transcription factors (TFs) in response to an environmental or intracellular stimulus. This code sets how each individual gene responds to stimuli by controlling when and where TFs bind. In my lab, our work is dedicated to developing a predictive understanding of gene expression. This is accomplished through a close interplay between quanti- tative theoretical predictions based on detailed biophysical models, and an experimental approach guided by falsifiable predictions for the quantitative consequence of systematic perturbation applied to a synthetic target gene. The work in my lab, which uses E. coli as a model organism, focuses on the systematic dissection of two distinct roles of regulatory binding sites in gene expression: The regulatory role of individual, local “cis-regulatory” binding sites. The regulatory role of non-local, competing binding sites scattered throughout the genome. To characterize local, cis-regulatory interactions we use a synthetic biology approach to systematically measure the regulatory function of every TF as a function of where on the gene it binds and to what sequence it binds. Through this process we will uncover the isolated function of each TF in the absence of the gene-specific factors (such as feedback and TF-TF interactions) that occlude this basic information in genomic data. To study the role of competing binding sites, we use this same synthetic biology approach to control the number and strength of TF binding sites to measure how competition for TFs controls spatial and temporal patterns in gene expression. Taken together, these directions of investigation are aimed at providing a complete picture of regulation at the level of a single gene. These methods are not orthogonal, it is difficult to study one without appreciating the other; to quantify how competition alters expression we must understand the “isolated” regulation of the local regulatory elements acting alone, and to study local regulatory elements we must understand the impact of the unavoidable, naturally occurring competing binding sites around the genome. Our approach is designed to isolate and quantify these regulatory effects to provide the foundation required to predict expression from the complex regulatory schemes observed in natural genes. In the next 5 years, we will demonstrate that by characterizing individual TF function based on binding location and sequence, we can disentangle the role of gene-specific features from basic TF function in order to under- stand how these components act together in the complex regulatory regions seen in naturally occurring genes. Furthermore, we will develop a theoretical model that accounts for the role of TF competition in orchestrating expression patterns in space and time that are crucial for cellular decision making and fitness.

项目概要/摘要 DNA中的调控密码规定了基因的表达将如何通过转录来调节 转录因子（TF）对环境或细胞内刺激的反应。这个密码设定了每个基因 通过控制转录因子结合的时间和地点来对刺激做出反应。在我的实验室里，我们致力于开发一种 对基因表达的预测性理解。这是通过数量之间的密切相互作用来实现的， 基于详细的生物物理模型的理论预测，以及由 对应用于合成目标的系统扰动的定量结果的可证伪预测 基因本实验室的工作是利用E.大肠杆菌作为模式生物，重点是系统解剖两个 调控结合位点在基因表达中的不同作用： 个体的局部“顺式调节”结合位点的调节作用。 分散在整个基因组中的非局部竞争结合位点的调节作用。 为了表征局部的顺式调节相互作用，我们使用合成生物学方法系统地测量 每一个TF的调节功能是它在基因上结合的位置和它结合的序列的函数。 通过这一过程，我们将揭示在缺乏基因特异性因子的情况下每个TF的独立功能。 (such作为反馈和TF-TF相互作用），其在基因组数据中封闭了该基本信息。研究这个角色 竞争结合位点，我们使用相同的合成生物学方法来控制竞争结合位点的数量和强度。 TF结合位点，以测量TF的竞争如何控制基因表达的空间和时间模式。 总的来说，这些调查方向的目的是提供一个完整的监管情况， 单个基因的水平。这些方法不是正交的，很难在不欣赏的情况下研究其中一种方法。 另一个;为了量化竞争如何改变表达，我们必须理解竞争的“孤立”调节。 局部调节因素单独作用，为了研究局部调节因素，我们必须了解其影响 基因组周围不可避免的、自然产生的竞争结合位点。我们的方法旨在 分离和量化这些调控效应，为预测表达提供基础。 在自然基因中观察到的复杂调控机制。 在接下来的5年里，我们将通过基于结合位置表征个体TF功能来证明， 和序列，我们可以从基本TF功能中分离出基因特异性特征的作用，以便了解 这些成分如何在天然存在的基因中的复杂调控区域中共同发挥作用。 此外，我们还将建立一个理论模型，解释TF竞争在协调中的作用 在空间和时间上的表达模式对细胞的决策和适应性至关重要。