# Regulation of transcription factor activity in neural crest development by pH dynamics

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2022 · $242,250

## Abstract

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...

## Key facts

- **NIH application ID:** 10508784
- **Project number:** 1R21DE032164-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** DIANE L BARBER
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $242,250
- **Award type:** 1
- **Project period:** 2022-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10508784

## Citation

> US National Institutes of Health, RePORTER application 10508784, Regulation of transcription factor activity in neural crest development by pH dynamics (1R21DE032164-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10508784. Licensed CC0.

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