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.
