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

Transcription factors (TFs) search for and bind specific DNA target sites to control gene expression. However, eukaryotic TF target search within the chromatinized genome is poorly characterized. Here, we combine in vivo and in vitro single-molecule imaging to dissect the role of differentially-charged DNA-binding domain flanking regions in the target search of Sox TFs. We demonstrate that the flanking region of Sox2 markedly enhances search efficiency compared to the negatively-charged flanking region of Sox17. The involved mechanisms are distinct for naked DNA compared to chromatin. On DNA, the enhanced search of Sox2 is driven by an increased target recognition rate during 1D sliding, despite reduced sliding speed. Conversely, enhanced nonspecific interactions between the Sox2’s DNA-binding domain flanking region and nucleosomes facilitate binding to compact chromatin and reinforce pioneer activity. These findings provide critical insights into biophysical mechanisms governing TF target search within the chromatinized genome. This work describes how positive charge in the peptide chain flanking the DNA-binding domain of transcription factors speeds up their search on free and chromatinized DNA, helping to explain why some transcription factors act as stronger pioneers.