Protein biophysics for pH-regulated transcription factor-DNA binding selectivity

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How transcription factors recognize specific DNA sequences to regulate gene expression remains a significant question in biology. This question is particularly perplexing when considering members of large families of transcription factors that share highly similar DNA- binding domains but bind selectively to different DNA sequences. Another significant question that remains understudied is how intracellular pH (pHi) dynamics regulate myriad cell behaviors, including proliferation, migration, differentiation and cell fate transitions. This project builds on prior studies of how pHi dynamics regulate cell behaviors via proteins whose activities are sensitive to changes in pHi. The research will explore the idea that pHi affects the DNA binding selectivity of transcription factors and thereby their function in regulating gene expression. The project also offers cross-disciplinary training opportunities from biophysics to cell biology for students and postdoctoral scholars.This project tests a new idea that pHi dynamics regulate transcription factor-DNA binding affinity and selectivity. At least 65 transcription factors in different families contain a conserved histidine in the DNA binding domain that forms hydrogen bonds with nucleotide bases. The hypothesis to be tested is that protonation and deprotonation of the histidine with changing pH regulates promoter binding selectivity of transcription factors, which is supported by molecular dynamics simulations and biochemical assays indicating pH modulates DNA binding by recombinant FOXM1 and FOXC2. The objectives include computational predictions of pH-dependent transcription factor binding to DNA (Aim 1), biochemical analysis of pH-dependent DNA binding affinities and motif preferences (Aim 2), and investigating the effects of pHi dynamics in transcription factor target gene selectivity in cells (Aim 3). The outcomes could reveal a new mechanism for pH-mediated regulation of transcription factor function, and offer new insights on how transcription factors with similar DNA-binding domains are used reiteratively to regulate a variety of genes.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.