Chromatin nanoscale compaction in live cells visualized by acceptor-donor ratio corrected FRET between DNA dyes

Abstract

Chromatin nanoscale architecture in live cells can be studied by Forster Resonance Energy Transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-donor ratio rather than distance. Here we show that a FRET value independent of the acceptor-donor ratio can be obtained by Fluorescence Lifetime Imaging (FLIM) detection of FRET combined with a normalization of the FRET level to a pixel-wise estimation of the acceptor-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that acceptor-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced micro-irradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.