Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84–97 events of 3D diffusion (3.3–3.7 s) interspersed with brief nonspecific collisions (0.75–0.9 s) before acquiring and dwelling at specific target DNA (12.0–14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters. [Display omitted] •Single-cell, single-molecule imaging shows Sox2/Oct4 dynamics in live ES cells•Sox2 locates target via a 3D diffusion-dominated search and 1D sliding along DNA•Sox2/Oct4 enhanceosome forms in a hierarchical binding order•Temporal patterns of target site occupancy are modulated by TF dynamics A single-cell, single-molecule approach provides a quantitative, real-time view of transcription factors’ search for target sites, revealing a 3D diffusion-dominated search, involving multiple collisions with nonspecific sites, as well as 1D sliding along DNA.