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Imaging Gene Regulation in Living Cells at the Single Molecule Level

Video   Jan 03, 2017

 

Combinatorial cis-regulatory network in animal genomes represents the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, it remains poorly understood how TFs dynamically search for specific target sites in the nucleus of a living cell and how the 3D spatial organization of the genome contributes to spatiotemporal gene expression programs during animal development. Here I will present our latest efforts in harnessing a set of advanced super-resolution, single-molecule imaging approaches to study how enhancer-binding pluripotency factors (Sox2/Oct4) dynamically search for and assemble on their cognate sites in live embryonic stem (ES) cells. Specifically, we developed a method capable of discriminating specific DNA binding events from non-specific macromolecular interactions in live cells; our single-molecule measurements also describe several key kinetic features (3D diffusion periods, number of trials, search times, residence times etc.) associated with in vivo TF target search dynamics and interestingly our data support an ordered Sox2-Oct4 assembly at endogenous loci. Finally, I will present an imaging strategy using the lattice light sheet microscope to probe nanometer-scale 3D enhancer organization in single live ES cells.

 
 
 
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