FOXC2-HIS122 and pH-dependent gene expression
Intracellular pH (pHi) dynamics regulates diverse cell processes such as proliferation, dysplasia, and differentiation, often mediated by the protonation state of a functionally critical histidine residue in endogenous pH sensing proteins. How pHi dynamics can directly regulate gene expression and whether transcription factors can function as pH sensors has received limited attention. We tested the prediction that transcription factors with a histidine in their DNA binding domain (DBD) that forms hydrogen bonds with nucleotides can have pH-regulated activity, which is relevant to more than 85 transcription factors in distinct families, including FOX, KLF, SOX and MITF/Myc. Focusing on FOX family transcription factors, we used unbiased SELEX-seq to identify pH-dependent DNA binding motif preferences, then confirm pH-regulated binding affinities for FOXC2, FOXM1, and FOXN1 to a canonical FkhP DNA motif that are 2.5 to 7.5 greater at pH 7.0 compared with pH 7.5. For FOXC2, we also find greater activity for an FkhP motif at lower pHi in cells and that pH-regulated binding and activity are dependent on a conserved histidine (His122) in the DBD. RNA-seq with FOXC2 also reveals pH-dependent differences in enriched promoter motifs. Our findings identify pH-regulated transcription factor-DNA binding selectivity with relevance to how pHi dynamics can regulate gene expression for myriad cell behaviours. Overall design: To investigate genes in which expression is dependent on FOXC2-His122 and pH, we established control (high pH) and clonal NHE1-KO (low pH) MDA-MB-436 cells. Next, we collected RNA from three biological replicates each from both control and NHE1-KO cells untransfected or transfected with FOXC2-WT, or pH-independent FOXC2 mutants H122K and H122N. To establish which genes are dependent on both FOXC2-His122 and pH, we filtered for genes that are only differentially expressed between control and NHE1-KO in the FOXC2-WT  condition but not untransfected or FOXC2- H122K, H122N.