The needle and the haystack: single molecule tracking to probe the transcription factor search in eukaryotes.

Transcription factors (TFs) regulate transcription of their target genes by identifying and binding to regulatory regions of the genome among billions of potential non-specific decoy sites, a task that is often presented as a 'needle in the haystack' challenge. The TF search process is now well understood in bacteria, but its characterization in eukaryotes needs to account for the complex organization of the nuclear environment. Here we review how live-cell single molecule tracking is starting to shed light on the TF search mechanism in the eukaryotic cell and we outline the future challenges to tackle in order to understand how nuclear organization modulates the TF search process in physiological and pathological conditions.

Chromatin organization drives the search mechanism of nuclear factors.

Nuclear factors rapidly scan the genome for their targets, but the role of nuclear organization in such search is uncharted. Here we analyzed how multiple factors explore chromatin, combining live-cell single-molecule tracking with multifocal structured illumination of DNA density. We find that factors displaying higher bound fractions sample DNA-dense regions more exhaustively. Focusing on the tumor-suppressor p53, we demonstrate that it searches for targets by alternating between rapid diffusion in the interchromatin compartment and compact sampling of chromatin dense regions. Efficient targeting requires balanced interactions with chromatin: fusing p53 with an exogenous intrinsically disordered region potentiates p53-mediated target gene activation at low concentrations, but leads to condensates at higher levels, derailing its search and downregulating transcription. Our findings highlight the role of disordered regions on factors search and showcase a powerful method to generate traffic maps of the eukaryotic nucleus to dissect how its organization guides nuclear factors action.