Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells

Cell (Impact Factor: 33.12). 03/2014; 156(6):1274-85. DOI: 10.1016/j.cell.2014.01.062
Source: PubMed

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.

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Available from: Zhengjian Zhang, Mar 21, 2014
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    • "Using eukaryotic gene regulation as an example, several SPT studies have reported the detection of transcription factor-DNA interaction kinetics in live cells (Gebhardt et al., 2013; Izeddin et al., 2014; Morisaki et al., 2014). In addition, a single-cell, single-molecule imaging method has been developed to systematically characterize the target search dynamics of the embryonic stem cell-specific transcription factors Sox2/Oct4 in live cells (Chen et al., 2014b). "
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    Molecular cell 05/2015; 58(4):644-659. DOI:10.1016/j.molcel.2015.02.033 · 14.46 Impact Factor
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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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    • "Interestingly, these proteins have dramatically longer residence times on DNA than GR based on published binding kinetics measured in vivo. GR (McNally et al., 2000; Mazza et al., 2012) and Sox2 (Chen et al., 2014) bind to DNA transiently in "
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