Electrostatic properties of disordered regions control transcription factor search and pioneer activity

Transcription factors (TFs) recognize specific target sites to control gene expression. While prokaryotic TFs combine 1D and 3D diffusion to efficiently locate their binding sites, the target search of eukaryotic TFs within the chromatinized genome is poorly characterized. Here we combine in vivo and in vitro single-molecule imaging to dissect the role of DBD-flanking regions (DFRs) of the mammalian Sox2 and Sox17 TFs in searching for their binding sites. We demonstrate that the DFR _Sox2 dramatically enhances the search efficiency of Sox2 as compared to the negatively-charged DFR _Sox17 . An enhanced search on naked DNA is primarily mediated by an increase in target recognition rate during 1D sliding. In contrast, target site recognition within nucleosomal DNA is mainly enhanced by increased nonspecific interactions with nucleosomes, facilitating binding to closed chromatin and reinforcing pioneering activity. These findings provide critical insights into the biophysical mechanisms governing TF target search on the chromatinized genome.