FOXC2 pH-dependent SELEX-seq

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. For SELEX-seq, a previously reported protocol (Riley et al. 2014) was used with modifications. We designed a library with a 16 base pair randomized region flanked by PCR sites GTTCAGAGTTCTACAGTCCGACGATCTGGNNNNNNNNNNNNNNNNTCGTATGCCGTCTTCTGCTTG. For each round of selective enrichment, final concentrations of 0.25 µM DNA library with 2.5 µM GST-FOXC2 were incubated for 30 min at RT in binding buffer (20 mM Hepes, 140 mM KCl, 0.05 mM TCEP-HCl) at either pH 7 or 7.8. Next, 30 µl of 50% pre-equilibrated glutathione Sepharose beads (Cytiva: 17075601) were added and samples were incubated end over end for 30 min at RT after which DNA-FOXC2-bead complexes were pelleted by centrifugation at 5000 rpm for 4 min. DNA-FOXC2-bead complexes were washed twice with 300 µl binding buffer and resuspended in 100 µl binding buffer prior to heat dissociation for 5 min at 95°C. Eluate DNA was clarified by centrifugation (10,000 rpm for 5 min) and PCR amplified with forward (SELEXF) and reverse (SELEXR) primers complementary to nucleotides flanking the randomized region. PCR products were cleaned using the MinElute PCR purification kit (Qiagen 28004) and the selection process was repeated four times with PCR products from prior rounds used as the starting library. 5’ and 3’ adapter overhangs were added by PCR to the initial starting library (R0) with all four rounds of selection (R1-4) used to prepare for addition of barcoded indexes. Indexes were added by PCR using the Nextera IDT UD Set D (Illumina 20027213) for multiplex sequencing