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bioRxiv · 10.1101/671784

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

Pelicci, S.·Diaspro, A.·Lanzano, L.
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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.

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Pelicci, S., Diaspro, A., Lanzano, L.. 2019-06-14. Chromatin nanoscale compaction in live cells visualized by acceptor-donor ratio corrected FRET between DNA dyes. https://doi.org/10.1101/671784

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