CAREER: Allosteric Regulation of Transcription Factor DNA Binding Specificity, Kinetics and Cellular Activity

职业：转录因子 DNA 结合特异性、动力学和细胞活性的变构调节

• 批准号: 1552862
• 负责人: Miles Pufall
• 金额: $ 116.24万
• 依托单位: University of Iowa
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1552862&HistoricalAwards=false
• 关键词: CAREER; Allosteric; Regulation; Transcription; Factor

Only a fraction of the over 20,000 genes present in every cell of the body are used at a given time. The choice of which genes to use is determined by environmental cues, such as heat, cold, food, hormones, and injury. This project studies how proteins function to turn genes on and off in response to hormones. Understanding this problem requires expertise from a variety of different disciplines, including physics, chemistry, and biology, that are often separate in the scientific culture. This research fosters cross-disciplinary training early, at the undergraduate level, by re-engineering how organic chemistry laboratory is taught, and connecting it to this pressing research problem. The results of this study provide critical insight into how cells respond to their environment while preparing the next generation of scientists to break down interdisciplinary barriers in the workplace.Proteins called transcription factors (TFs) have the critical task of integrating intra- and extracellular signaling pathways to precisely regulate gene expression in response to stimuli. TFs regulate genes by binding specific DNA sequences and nucleating the machinery needed for transcription (RNA synthesis). Recent large-scale efforts have defined the core DNA sequence preferences for hundreds of TFs, but very little is known about how these crucial regulatory signals change TF:DNA binding activity. This project may determine how external signals remodel the intrinsic properties of TF:DNA binding to redirect genomic association and gene regulation. Using the glucocorticoid receptor (GR, a hormone activated TF) as a model system, the effect of ligands and phosphorylation on the specificity and kinetics of DNA binding are measured in vitro, and compared to GR genomic occupancy and DNA binding dynamics in live cells. DNA binding specificity are measured in vitro using high resolution systematic evolution of ligands by exponential enrichment (SELEX-seq) from which thermodynamic models of binding covering all possible sequences are generated, and in cells using chromatin-immunoprecipitation followed by deep sequencing (ChIP-seq). DNA binding kinetics are measured in vitro using single molecule total internal reflection fluorescence microscopy (TIRFM) and in cells using single molecule single molecule tracking (SMT). The consequence of changing specificity and kinetics are then tested using cell lines with engineered binding sites generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats). Students in the undergraduate Advanced Organic Laboratory course at Butler University synthesize some of the ligands to be used in the research, and also participate in their testing. The results build on current static descriptions for understanding specificity and regulation, by enabling creation of new models that predict the effect of changing intrinsic properties on regulating cellular function.This project is co-funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences and the Division of Emerging Frontiers in the Biological Sciences Directorate, and by the Chemistry of Life Processes Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.

在身体的每个细胞中存在的超过20，000个基因中，只有一小部分在给定的时间内被使用。选择使用哪些基因取决于环境因素，如热、冷、食物、激素和伤害。该项目研究蛋白质如何在响应激素的情况下打开和关闭基因。理解这个问题需要来自各种不同学科的专业知识，包括物理学，化学和生物学，这些学科在科学文化中通常是独立的。这项研究促进跨学科的培训早期，在本科阶段，通过重新设计有机化学实验室的教学方式，并将其连接到这个紧迫的研究问题。这项研究的结果提供了关键的洞察细胞如何响应他们的环境，同时准备下一代科学家打破跨学科的障碍，在工作场所.蛋白质称为转录因子（TF）的关键任务整合内和细胞外信号通路，以精确调节基因表达响应刺激.转录因子通过结合特定的DNA序列和使转录所需的机制（RNA合成）成核来调节基因。最近的大规模努力已经确定了数百个TF的核心DNA序列偏好，但对这些关键的调控信号如何改变TF：DNA结合活性知之甚少。该项目可能会确定外部信号如何重塑TF的内在特性：DNA结合重定向基因组关联和基因调控。使用糖皮质激素受体（GR，一种激素激活的TF）作为模型系统，在体外测量配体和磷酸化对DNA结合的特异性和动力学的影响，并与活细胞中的GR基因组占有率和DNA结合动力学进行比较。DNA结合特异性在体外使用通过指数富集的配体的高分辨率系统进化（SELEX-seq）来测量，其中从所述配体的高分辨率系统进化产生覆盖所有可能序列的结合的热力学模型，并且在细胞中使用染色质免疫沉淀随后进行深度测序（ChIP-seq）来测量。使用单分子全内反射荧光显微镜（TIRFM）在体外和使用单分子单分子跟踪（SMT）在细胞中测量DNA结合动力学。然后使用具有使用CRISPR/Cas9（成簇的规则间隔的短回文重复序列）生成的工程化结合位点的细胞系来测试改变特异性和动力学的结果。巴特勒大学本科高级有机实验室课程的学生合成了一些用于研究的配体，并参加了他们的测试。 这些结果建立在当前理解特异性和调节的静态描述的基础上，通过创建新的模型来预测改变内在特性对调节细胞功能的影响。该项目由分子和细胞生物科学部的遗传机制计划和生物科学理事会的新兴前沿部门共同资助，以及数学和物理科学理事会化学部的生命过程化学计划。