title: Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.
abstract: 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.
authors.affiliation: Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Jiji
authors.initials: J
authors.lastname: Chen
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Zhengjian
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Bi-Chang
authors.initials: BC
authors.lastname: Chen
authors.affiliation: Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Andrey
authors.initials: A
authors.lastname: Revyakin
authors.affiliation: Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA; Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Paris 75794, France.
authors.forename: Bassam
authors.initials: B
authors.lastname: Hajj
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Wesley
authors.initials: W
authors.lastname: Legant
authors.affiliation: Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA; Laboratoire Physico-Chimie Curie, Institut Curie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Paris 75794, France.
authors.forename: Maxime
authors.initials: M
authors.lastname: Dahan
authors.affiliation: Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Timothée
authors.initials: T
authors.lastname: Lionnet
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Betzig
authors.affiliation: Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA; Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA; LKS Bio-Medical and Health Sciences Center, CIRM Center of Excellence, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Tjian
authors.affiliation: Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA; Junior Fellow Program, Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA. Electronic address: liuz11@janelia.hhmi.org.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D044127:Epigenesis, Genetic; D055106:Genome-Wide Association Study; D007700:Kinetics; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism*; D059010:Single-Cell Analysis
chemical_list: D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D004247:DNA