Abstract Our proposal tests a new idea on how transcription factor-DNA binding selectivity is regulated within the context of neural crest (NC) development and intracellular pH (pHi) dynamics. Transcription factors in FOX, SOX, MITF, MYC, and other families with established roles in NC development and craniofacial lineages have a conserved histidine that forms hydrogen bonds with DNA nucleotides. With the ability of histidine to titrate within the cellular pH range and nuclear and cytosolic pH values being similar, our idea proposes that histidine-nucleotide binding affinities (Kd’s) and hence promotor selectivity can be regulated by pHi dynamics. Despite this idea being applicable to at least 65 transcription factors across multiple families it has largely escaped the notice of investigators across different fields. The biophysical principles of our prediction are that when histidine is protonated at a lower pH it will be a hydrogen bond donor with a hydrogen bond acceptor nucleotide, such as thymine, and when histidine is deprotonated at a higher pH it will be a hydrogen bond acceptor with a hydrogen bond donor nucleotide, such as adenine. Hence, we will test the hypothesis that pHi dynamics regulates transcription factor-DNA binding selectivity for neural crest development by focusing on three transcription factors from different families, FOXD3, SOX10, and MITF, that have established roles in NC development and specification of craniofacial lineages. Our hypothesis is supported by pHi dynamics regulating gene expression for stem cell differentiation and lineage specification, abundant structure data, and our preliminary findings. Moreover, our hypothesis addresses a critical gap in our understanding of how transcription factors are used reiteratively in developmental programs. Contributing to the success of our proposal is our work pioneering a molecular understanding of how pHi dynamics regulates myriad cell behaviors by bridging protein electrostatics and cell biology. In Aim 1 we will determine pH-dependent DNA binding affinities and motif preferences for FOXD3, SOX10 and MITF. We will determine pH regulated Kd’s of recombinant DNA binding domains to previously identified motifs by using fluorescence anisotropy, supported by preliminary data on pH regulated binding affinities of FOXM1 and FOXC2, and identify pH-dependent genome-wide binding preferences with the unbiased approach of systemic evolution of ligand by exponential enrichment (SELEX). In Aim 2 we will determine the role of pHi dynamics in transcription factor-DNA binding in iPSC-derived neural crest cells and in zebrafish models. Cell studies will identify pHi regulated motif preferences by using a dual fluorescent reporter we developed and by ChIP-seq. Zebrafish studies, supported by our data showing spatial differences in pHi in zebrafish embryos during the period of NC development, will test rescue of defects with homozygous- null sox10 and mitf. If our predictions are correct, the outcome of our proposal would be identifying for the first time that transcription factors can be pH sensors with pHi regulating promoter selectivity for NC development, with our findings demonstrating in molecular detail how this occurs and with relevance to craniofacial anomalies.

Project Narrative Our proposal tests a new idea on how changes in intracellular pHi directly regulate gene expression through titration of a histidine in the DNA-binding domain of transcription factors. We will test our idea by focusing on transcription factors regulating neural crest development by using biochemistry of recombinant proteins, the biology of clonal neural crest cells, and zebrafish embryos. Our findings are highly relevant to understanding human malformations from defects in neural crest development, including myriad craniofacial anomalies.