Systematic determination of a transcription factor/binding site functional interaction landscape

Abstract

Cells require their genetic information to be expressed appropriately; the ability to modulate gene expression in a proper spatiotemporal response to external and internal signals is central to survival. Transcription factors are a major class of regulatory proteins that specifically bind DNA to modulate the expression of targeted genes. While they have been extensively studied, major questions remain about the protein-DNA interaction underlying this hub of regulation. What binding site sequences functionally interact with a given regulator? How does the regulon sample from available functional sequences? How independent is each half of a two part binding site? How do mutations in the regulator impact the regulon? Using PhoP, the regulator from the E. coli magnesium-responsive two-component system PhoPQ, I sought to address these questions. I identified the genomic binding locations for PhoP, verifying and expanding our knowledge of the PhoP regulon. Using two randomized libraries of over 65,000 variants each, I interrogated how changes in DNA sequence impact functional binding of PhoP. Comparing this with genomic binding data showed PhoP regulon members may avoid some sequences based on the dysfunctionality of their neighboring sequences. The functional library sequences reveal context dependence for each half-site and interaction within and across binding site halves. Finally, using an orthogonal PhoP mutant, I found that although these two proteins interacted with very few overlapping promiscuous sequences, there were many single mutations that would switch a promoter from interacting specifically with one protein to the other.