Observing protein interaction dynamics to chemically defined chromatin fibers by colocalization single-molecule fluorescence microscopy

In eukaryotic cells, the genome is packaged into chromatin and exists in different states, ranging from open euchromatic regions to highly condensed heterochromatic regions. Chromatin states are highly dynamic and are organized by an interplay of histone post-translational modifications and effector proteins, both of which are central in the regulation of gene expression. For this, chromatin effector proteins must first search the nucleus for their targets, before binding and performing their role. A key question is how chromatin effector proteins search, interact with and alter the different chromatin environments. Here we present a modular fluorescence based in vitro workflow to directly observe dynamic interactions of effector proteins with defined chromatin fibres, replicating different chromatin states. We discuss the design and creation of chromatin assemblies, the synthesis of modified histones, the fabrication of microchannels and the approach to data acquisition and analysis. All of this with the aim to better understand the complex in vivo relationship between chromatin structure and gene expression.