A role for pH dynamics regulating transcription factor DNA-binding selectivity

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Intracellular pH (pHi) dynamics regulate critical cellular processes like proliferation and differentiation.
• The role of pHi in directly controlling gene expression via transcription factor activity remains under-explored.
• Histidine residues in pH-sensing proteins are key mediators of pHi-dependent cellular functions.

Purpose of the Study:

• To investigate whether transcription factors can act as pH sensors, directly regulating gene expression.
• To test the hypothesis that histidine residues in transcription factor DNA-binding domains (DBDs) mediate pH-regulated DNA binding.
• To explore the impact of pHi dynamics on transcription factor binding specificity and cellular behavior.

Main Methods:

• Systematic Evolution of Ligands by Exponential Enrichment followed by Sequencing (SELEX-seq) to identify pH-dependent DNA-binding motifs.
• Electrophoretic mobility shift assays (EMSAs) to confirm pH-regulated binding affinities.
• Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to analyze genome-wide binding and gene expression changes.
• Site-directed mutagenesis to assess the role of specific histidine residues.

Main Results:

• Identified pH-dependent DNA-binding motif preferences for FOX family transcription factors (e.g., FOXC2, FOXM1, FOXN1).
• Demonstrated that binding affinities for FOXC2, FOXM1, and FOXN1 to specific motifs are significantly altered by changes in pHi (pH 7.0 vs. 7.5).
• Showed that the pH-dependent activity of FOXC2 is mediated by a conserved histidine (His122) in its DBD.
• ChIP-seq and RNA-seq revealed pH-dependent differences in FOXC2 promoter enrichment and gene expression.

Conclusions:

• Transcription factors with histidine residues in their DBDs can function as direct pH sensors, exhibiting pH-regulated DNA-binding selectivity.
• pHi dynamics can directly modulate transcription factor activity, influencing gene expression and cellular behaviors.
• This mechanism is relevant to over 85 transcription factors across multiple families, highlighting a fundamental regulatory pathway in cell biology.