Overlapping binding sites underlie TF genomic occupancy

Abstract Sequence-specific DNA binding by transcription factors (TFs) is a crucial step in gene regulation. However, current high-throughput in vitro approaches cannot reliably detect lower affinity TF-DNA interactions, which play key roles in gene regulation. Here, we developed PADIT-seq (protein affinity to DNA by in vitro transcription and RNA sequencing) to assay TF binding preferences to all 10-bp DNA sequences at far greater sensitivity than prior approaches. The expanded catalogs of low affinity DNA binding sites for the human TFs HOXD13 and EGR1 revealed that nucleotides flanking high affinity DNA binding sites create overlapping lower affinity sites that together modulate TF genomic occupancy in vivo. Formation of such extended recognition sequences stems from an inherent property of TF binding sites to interweave each other and expands the genomic sequence space for identifying noncoding variants that directly alter TF binding. One-Sentence SummaryOverlapping DNA binding sites underlie TF genomic occupancy through their inherent propensity to interweave each other.