Free Energy Spectroscopy, a new method for investigating chromatin compaction.

The physical organization of eukaryotic genomes is evolutionarily conserved, where histone protein octamers repeatedly wrap genomic DNA into nanometer-sized nucleosome spools to form chromatin: the basic organization structure of all eukaryotic genomes. The chromatin state of compaction is critical for the regulation of gene expression, where compact heterochromatin suppresses gene expression while open euchromatin activates it. However, the mechanisms of regulation and maintenance of chromatin states remain poorly understood. The relative probability of open and compact chromatin states is determined by the free energy differences between each state through the Boltzmann distribution. So, determining the relative free energies of chromatin structural conformations is key to understanding the relative probability differences between different euchromatin and heterochromatin states, which is central for understanding eukaryotic genome accessibility and ultimately function. We have developed a new method for quantifying chromatin compaction, Free Energy Spectroscopy (FES), which is based on DNA nanotechnology and transmission electron microscopy. This new method allows us to map out multidimensional free energy landscapes of chromatin compaction, which, to date, has only been investigated with computational methods. We have applied this FES method to understand the regulation of chromatin compaction by (i) linker histones, which are ubiquitous chromatin organization proteins that generate compact heterochromatin and suppress genome accessibility to transcription regulatory factors, and (ii) transcription factors, which target specific DNA sequences to initiate chromatin decompaction to then activate transcription. Furthermore, the success of these studies suggests that FES is an impactful tool to help answer a broad range of mechanistic questions about genome and epigenome function in the test tube and potentially even in live cells.