Impact of DNA sequence on the structure and dynamics of the higher-order chromatin fibre in vitro

Abstract

DNA in the eukaryotic cell is organised into a complex called chromatin, which protects the DNA from damage and allows the careful regulation of the genome. Precisely how the genome is used by the cell is dependent on the structure and regulation of this complex, and discovering how this is organised is therefore one of the principle challenges of modern biology. DNA is wrapped around histone octamers to form arrays of nucleosomes, which are subsequently folded into a higher-order structure, speculated to be a 30-nm fibre. In vitro studies of higher-order chromatin fibre structure have provided valuable information about this structure, but it is not well understood how changes to the DNA sequence might affect the structure and dynamics of the complex. DNA sequence is known to affect nucleosome binding strength and positioning within an array, and I therefore hypothesised that DNA sequence changes are likely to impact higher-order chromatin fibre structure. Using an in vitro model of chromatin fibre structure, reconstituting purified DNA with purified core histones by salt dialysis, allowed me to isolate the effects of DNA sequence in the absence of confounding factors such as transcription factor binding. I compared the higher-order chromatin structure and dynamics of the well-studied “601” DNA repeat sequence with two novel reconstitution templates which contain biologically-derived nucleosome positioning sequences. Sucrose gradient sedimentation of folded chromatin fibres suggested that non-601 fibres may be as compacted as 601 fibres, but have more heterogeneous structures. However, non-601 fibres were more easily perturbed under tension than 601 fibres, suggesting that such sequences might promote a more accessible chromatin environment. While repetitive 601 fibres were found to have a regular nucleosome repeat length by DFF digestion, non-repetitive, biologically-derived sequences had a more heterogeneous nucleosome repeat length, which I suggest is responsible for their increased accessibility. I also found that the compacted higher-order structure of the 601 fibre is disrupted by the introduction of a single sequence with low affinity for the histone octamer. These structures can be separated by sucrose gradient sedimentation, and I suggest that this could be a useful method to examine the individual effects of a wider range of DNA sequences on higher-order chromatin fibre structure in vitro.