Multiple overlapping binding sites determine transcription factor occupancy.

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Transcription factors (TFs) regulate gene expression through sequence-specific DNA interactions.
• Existing high-throughput methods struggle to identify low-affinity TF binding sites, crucial for gene regulation.
• Low-affinity sites are increasingly recognized for their role in precise spatiotemporal gene expression control.

Purpose of the Study:

• To develop a novel method for comprehensively assaying TF DNA-binding preferences.
• To identify previously undetected low-affinity TF binding sites.
• To propose a new model for TF binding and its role in gene regulation and disease.

Main Methods:

• Development of protein affinity to DNA by in vitro transcription and RNA sequencing (PADIT-seq).
• Comprehensive assay of binding preferences for six TFs across all ten-base-pair DNA sequences.
• Analysis of TF binding site overlap and its impact on genomic occupancy.

Main Results:

• PADIT-seq successfully detected hundreds of novel, lower-affinity DNA binding sites for TFs.
• Nucleotides flanking high-affinity sites create overlapping lower-affinity sites that modulate in vivo TF binding.
• A model of TF binding based on the sum of multiple overlapping sites was proposed.

Conclusions:

• TF binding is determined by the collective effect of multiple, overlapping binding sites, not just individual high-affinity sites.
• The overlapping binding model explains TF competition and differential binding site usage by paralogous TFs.
• This model redefines the impact of noncoding variants, showing how they alter multiple sites to influence gene expression, human traits, and diseases.