DNA flexibility regulates transcription factor binding to nucleosomes

Cell fate decisions are controlled by sequence-specific transcription factors (TFs), referred to as 'pioneer' factors, that bind their target sites within nucleosomes ('pioneer binding') and thus initiate chromatin opening. However, pioneers bind just a minority of their recognition sequences present in the genome, suggesting that local sequence context features may regulate pioneer binding. Here, we developed PIONEAR-seq, a highly parallel sequencing-based biochemical assay for high-throughput analysis of TF binding to nucleosomes on nucleosome positioning sequences. Using PIONEAR-seq, we characterized the pioneer binding of 7 human pioneer TFs. Comparison of TF binding to nucleosomes based on the synthetic Widom 601 (W601) model sequence versus three different genomic sequences revealed that the positional preferences of these TFs' binding to nucleosomes (i.e., dyad, periodic and end binding) is determined by the broader sequence context of the nucleosome, rather than being a property intrinsic to the TF. We propose a model where the flexibility and rigidity within nucleosomal DNA regulate where pioneers bind within nucleosomes. Our results suggest that the broader physical properties of nucleosomal DNA represent another layer of cis-regulatory information read out by TFs in eukaryotic genomes.