Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells

Cell (Impact Factor: 32.24). 03/2014; 156(6):1274-85. DOI: 10.1016/j.cell.2014.01.062
Source: PubMed


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.

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Available from: Zhengjian Zhang, Mar 21, 2014
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    • "The thermodynamic modeling approach has its own limitation. For example, it does not take into account the dynamical effects of the enhanceosome assembly[25,26], which might influence the relative probabilities of different molecular configurations of the regulatory regions. The implemented methods for TFBS search may also contain errors, leading to possible mispredictions for binding sites. "
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    • "In vivo analysis of the binding regimes of transcription factors shows that some use facilitated diffusion (i.e., 3D diffusion interspersed with local 1D sliding along the chromatin fiber). This greatly reduces their search space to give an efficient means of finding their specific binding sites (Chen et al. 2014;Izeddin et al. 2014;Woringer et al. 2014;Normanno et al. 2015). A transcription factor bound at an enhancer might then rather efficiently, by oversampling the local volume, be able to find a promoter-proximal binding site locatedwithin the same chromatin domain, obviating any need for a specific enhancer– promoter chromatin loop (Figs. "
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    • "Yet experimental data also shows that TF binding to DNA can alter chromatin structure [32] [49] [67], which is also relevant as to how gene expression is regulated [83]. But perhaps more intriguing in this context are the experiments of single molecule tracking of (animal ) TFs, which show how TF molecules move across chromatin in their search for their target genes [84] [85]. These TF search events "
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