Transcription factor genomic occupancy is determined by multiple, overlapping DNA binding sites
Transcription factors (TFs) regulate gene expression by interacting with DNA in a sequence-specific manner. High-throughput in vitro technologies, such as PBMs and HT-SELEX, have revealed the DNA binding specificities of hundreds of TFs. However, they have limited ability to reliably identify lower affinity binding sites, which are increasingly recognized as important for precise spatial and temporal control of gene expression. To address this limitation, we developed a novel technology to measure protein affinity to DNA by in vitro transcription and RNA sequencing (PADIT-seq). Using PADIT-seq, we comprehensively assayed the binding preferences of four human and two yeast TFs to all possible 10-bp DNA sequences, detecting hundreds of novel lower affinity binding sites. The expanded repertoire of lower affinity binding sites unexpectedly revealed that nucleotides flanking high affinity DNA binding sites create overlapping lower affinity sites that together modulate TF genomic occupancy in vivo. We propose a model where TF binding is not determined by individual binding sites, but rather by the sum   of multiple, overlapping binding sites. Formation of such extended recognition sequences stems from an inherent property of TF binding sites to interweave each other. Consequently, competition between paralogous TFs for shared high-affinity binding sites is controlled by flanking nucleotides that create differential numbers of overlapping lower affinity binding sites. Noncoding variants alter multiple, overlapping binding sites to influence gene expression and human traits, including diseases. Overall design: Binding preferences of 6 TFs to all possible 10-mers using PADIT-seq, a novel technology to detect TF-DNA interactions across the entire spectrum of affinities.