EAGER: A high-throughput in vivo method for measuring transcription factor-DNA interactions

被称为转录因子的蛋白质和DNA之间的相互作用是细胞功能的核心。这些相互作用是特定的和物理的--蛋白质和DNA分子在物理上相互作用，如果特定的蛋白质和DNA序列兼容，这种相互作用就会持续足够长的时间，以至于复杂的分子机器可以被招募到那个位置来制造新的蛋白质。测量这种相互作用是了解疾病、对生物体进行基因工程、预测进化和破译发育如何受到调控的努力的关键组成部分。该项目将开发一种新的方法来大规模测量和分析这些蛋白质-DNA相互作用，在一个实验中一次性检查数百万个DNA变体和蛋白质变体之间的相互作用。这种方法使用了广泛可用的资源，因此它应该使这些类型的测量对大量的研究人员、学生和实习生来说是可行的，包括那些在研究基础设施不大的机构的人。这个项目的基础是开发和测试一个新的测量蛋白质-DNA相互作用的平台。该分析的设计使得新的蛋白质或DNA片段可以很容易地插入平台，快速产生用于分析的巨大蛋白质-DNA变异库。与现有用于测量这些相互作用的技术不同，这种分析简化了蛋白质生产的过程，因此可以测试环境条件甚至其他蛋白质的存在是否会改变结合的强度。作为其潜力的展示，该方法将确认和扩展先前关于人类HoxD13基因的多态如何改变其与其目标结合位点的结合程度的研究，并将用于探索一组密切相关和共同表达的GATA型转录因子在线虫属进化过程中的结合偏好如何多样化。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

Interactions between proteins called transcription factors and DNA lie at the very heart of how a cell functions. These interactions are specific and physical - proteins and DNA molecules physically interact and if a particular protein and a DNA sequence are compatible then the interaction persists long enough for complex molecular machinery to be recruited to that location to make new proteins. Measuring this interaction is a crucial component in efforts to understand disease, genetically engineer organisms, predict evolution, and decipher how development is regulated. This project will develop a new method for measuring and analyzing these protein-DNA interactions at a massive scale, examining millions of interactions between DNA variants and protein variants all at once in a single experiment. This method uses resources that are widely available and so it should make these kinds of measurements feasible for great number of researchers, students, and trainees, including those at institutions with modest research infrastructure.The basis of this project is development and testing of a new platform for measuring protein-DNA interactions. The assay is designed so that new proteins or stretches of DNA can easily be plugged into the platform, quickly producing enormous libraries of protein-DNA variants for analysis. Unlike existing technologies that are used to measure these interactions, this assay simplifies the process of protein production so that it is possible to test whether environmental conditions or even the presence of other proteins change the strength of the binding. As a demonstration of its potential, the method will confirm and extend previous studies of how polymorphisms in human HoxD13 change how well it binds to its target binding sites, and it will be used to explore how the binding preferences of a group of closely related and co-expressed GATA-type transcription factors diversified during the evolution of the Caenorhabditis genus.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.