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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    • "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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    • "This can be rationalized by the dynamic association of TFs with DNA, the limited occupancy times for TFs, and the ability of TFs to bring in different co-activator complexes. In line with this, photo-bleaching experiments (McNally et al. 2000; Mueller et al. 2008; Bosisio et al. 2006) and single molecule tracking studies (Chen et al. 2014; Gebhardt et al. 2013) showed that the average residence time of TFs on DNA is in the range of seconds to minutes. Interestingly, it has been shown that the residence time of a TF at a single site varies and that longer interactions correlate with higher transcriptional output (Lickwar et al. 2012; Karpova et al. 2008; Stavreva et al. 2004; Sharp et al. 2006) (Fig. 2). "
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