Defining transcription factor nucleosome binding with Pioneer-seq

The majority of Eukaryotic DNA is wrapped into nucleosomes that is largely resistant to transcription factor (TF) binding. This inherent inability to specifically bind to nucleosomal DNA creates a conundrum during development as many gene regulatory regions are inaccessible. The rules dictating TF nucleosome binding remain largely unknown. To comprehensively examine TF nucleosome binding we developed Pioneer-Seq (transcriPtIon factOr NuclEosomE binding pRotocol). For Pioneer-seq 7500 in vitro nucleosomes are formed from multiple DNA sequences containing a variant of the TF binding site (TFBS) in a unique nucleosome orientation. These nucleosomes are then bound to the TF of interest and the protein-nucleosome complex is isolated and the nucleosome sequence determined by next-generation sequencing. Pioneer-seq can examine nucleosomes created with various nucleosome positioning sequences (NPS) and examine binding to in vivo targeted nucleosomes (ITNs). TFBSs can be tiled at every translational and rotation setting with various TFBS examined in the same assay. To demonstrate Pioneer-seq we examine nucleosome binding by OCT4, SOX2, KLF4, and c-MYC. Our results demonstrate that each TF has differing binding capabilities to nucleosomal DNA with nucleosome sequence being a major factor regulating TF binding. Furthermore, we show that KLF4 and SOX2 can bind close to the nucleosome dyad at specific rotational settings with examination of ITNs showing similar results.