Regulation of large-scale chromatin architecture in mammalian cells

Abstract
Chromatin is an extremely large heterogenous macromolecule with many different levels of folding. This presents a number of challenges in studying its architecture and necessitating the development of new experimental approaches. In one of our previous studies we showed a relationship between large-scale chromatin structures (100s kb) and transcription. On the surface this result seemed surprising – an event occurring at the level of a promoter/gene impacting on large-scale structures. From this observation we reasoned there must be some property that can link different levels of genome organization from the level of a gene (10’s kb) with higher levels of genome structure (100’s kb). One of the assays we use routinely to investigate chromatin structure is to measure the distance between pairs of FISH probes, hybridized to interphase nuclei. A surprising result came when we treated cells with the drug bleomycin; bleomycin is an antibiotic that introduces single stranded breaks into the chromatin. After bleomycin treatment we measured the distance between our FISH probes and observed a significant change in large-scale chromatin structure. This enabled us to hypothesise that large scale chromatin fibres were under topological strain and we predicted that supercoiling generated by transcription could be propagated along the chromatin fibre affecting large scale chromatin structures. I will present the experiments we used to test this idea. I will also show new data that reveals a mechanism for remodelling large-scale chromatin structure, via chromatin associated RNAs, and shows that interphase chromatin architecture is necessary to prevent aberrant mitotic chromosome structures and genome instability.