pH dynamics determining DNA binding specificity of FOX transcription factors

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Project Summary/Abstract Members of the mammalian FOX family of transcription factors regulate a broad range of cellular processes including cell division, differentiation, and apoptosis. Despite sharing a highly conserved DNA-binding sequence, each FOX family member binds a distinct set of target genes to regulate myriad cell behaviors. The current understanding of DNA-binding selectivity of different FOX family transcription factors is that regulation is conferred by co-factor association, post-translational modifications, and cell specific expression. This proposal aims to resolve a new idea that intracellular pH (pHi) dynamics is an additional mechanism regulating target gene selectivity of FOX family transcription factors. Although all FOX proteins contain an invariant histidine residue in the DNA-binding domain that directly forms hydrogen bonds with nucleotides and histidine residues can titrate with pHi dynamics, whether pHi regulates nucleotide binding specificity of FOX proteins has not been reported. Building on preliminary data demonstrating pH-dependent binding of FoxM1 to a common consensus sequence, this proposal tests the overall hypothesize that the invariant histidine in FOX DNA-binding domains is a pH sensor for pHi-regulated binding selectivity to target genes. The objective of Aim 1 is to determine pH-regulated DNA binding selectivity of FOX family proteins. Predictions on pH-regulated DNA- binding specificity of a number of FOX family members will be tested in silico using molecular dynamic simulations that will inform in vitro biochemical approaches to determine binding affinities using fluorescence anisotropy. Additionally, genome-wide pH-dependent binding preferences will be determined using systematic evolution of ligand by exponential enrichment (SELEX). The objective of Aim 2 is to determine the role of pHi dynamics in regulating FoxM1 target genes in cells, focusing on naïve mouse embryonic stem cells and cancer cells because they to have behaviors regulated by pHi dynamics. Global FoxM1 promoter binding preferences in response to pHi dynamics will be identified in embryonic stem cells and cancer cells by using ChiP-seq that will inform mechanistic analysis by determining pHi regulated gene expression using RT-qPCR. In addition to revealing a new paradigm for transcriptional regulation of FOX proteins the principles being tested have significance for the activity of other transcription factors that contain histidines in the DNA binding domain, including members of IRF, GATA and ETV families. Moreover, because pHi-regulated transcription factor activity and gene expression remain understudied, outcomes will add mechanistic understanding of how pHi dynamics regulates cell behaviors. Finally, because FOX transcriptional activity and pHi dynamics are dysregulated in many diseases, these findings will have clinical relevance toward new therapeutic approaches, particularly to limit cancer progression and also for controlling regenerative medicine.