Transcription factors (TFs) are essential regulators of gene expression. TFs locate their target sites by combining 1D sliding along DNA with 3D diffusion in the nuclear environment. Although this mechanism has been thoroughly explored in prokaryotes, the behavior of TFs in eukaryotic cells, where DNA is compacted into chromatin, is not as well understood. In particular, recent studies have indicated a role of intrinsically disordered regions (IDRs) adjacent to the DNA-binding domains (DBDs) of eukaryotic TFs in the target search process.
On the other hand, TFs sharing the same DBDs do not bind to the same sites on the genome. Considering that TF-DNA nonspecific interactions mediated by electrostatic forces facilitate the target search, we aim to uncover how charged regions flanking the DBDs impact TF target search and binding efficiency. To this end, we designed a comparative study between the two Sox TFs, Sox2 and Sox17. Both bind to the same DNA motif, but Sox2 has positively charged flanking regions, while Sox17 has a more negatively charged IDR.
We employed both in vivo and in vitro single-molecule tracking (SMT) analysis to study TF target search efficiency. In vivo SMT in mouse embryonic stem cells showed that Sox2, with a basic stretch, had higher association rates than Sox17 and Sox2a, a mutant with an acidic stretch of Sox17. In vitro SMT confirmed that Sox2 exhibited enhanced target search with both naked DNA and chromatin.
Further analysis revealed that Sox2 had a 2-fold higher specific on-rate through 1D sliding compared to Sox2a. Additionally, Sox2 exhibited a 3-fold higher nonspecific on-rate to chromatin. Our results suggest that positively charged IDRs enhance the target search by facilitating 3D nonspecific interactions with chromatin, which promotes chromatin invasion, and accelerates the specific target recognition on bare DNA.
Overall, this study demonstrates a biophysical mechanism underlying the target search process of eukaryotic TFs, showing that positively charged IDRs are crucial for efficient target localization within chromatin. Our findings offer insights into the fundamental processes that enable TFs to identify their specific targets within the densely packed chromatin of eukaryotic cells, thereby advancing our understanding of gene regulation in complex cellular environments.