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

在给定的时间内，人体每个细胞中存在的20,000多个基因中只有一小部分被使用。选择使用哪种基因取决于环境提示，如热、冷、食物、激素和伤害。这个项目研究了蛋白质如何在荷尔蒙的作用下开启和关闭基因。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、化学和生物学，这些学科在科学文化中往往是分开的。这项研究通过重新设计有机化学实验室的教学方式，并将其与这一紧迫的研究问题联系起来，早期地在本科水平上促进了跨学科培训。这项研究的结果为细胞如何响应环境提供了关键的见解，同时为下一代科学家打破工作场所的跨学科障碍做好准备。被称为转录因子(TF)的蛋白质具有整合细胞内和细胞外信号通路的关键任务，以精确调节基因表达以响应刺激。转录因子通过结合特定的DNA序列和使转录(RNA合成)所需的机制成核来调节基因。最近的大规模研究已经确定了数百个转录因子的核心DNA序列偏好，但对这些关键调控信号如何改变TF：DNA结合活性知之甚少。这个项目可能确定外部信号如何重塑TF：DNA结合的内在属性，以重定向基因组关联和基因调控。以糖皮质激素受体(GR，一种激素激活的转铁蛋白)为模型系统，在体外测定了配体和磷酸化对DNA结合的特异性和动力学的影响，并与GR基因组占有率和活细胞中DNA结合动力学进行了比较。DNA结合的特异性在体外使用高分辨率的指数富集型配体系统进化(SELEX-SEQ)来测量，由此产生覆盖所有可能序列的结合热力学模型，并在细胞中使用染色质免疫沉淀和深度测序(CHIP-SEQ)。使用单分子全内反射荧光显微镜(TIRFM)和单分子单分子跟踪(SMT)技术分别在体外和细胞内测量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)产生的工程结合位点的细胞系来测试改变的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们合成了一些用于研究的配体，并参与了它们的测试。这个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局新兴前沿部门的遗传机制计划以及数学和物理科学局化学部的生命过程化学计划共同资助。

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.