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Published online
doi:10.1083/jcb.200907029
The Journal of Cell Biology, Vol. 187, No. 4, 481-496
The Rockefeller University Press, 0021-9525 $30.00
© Llères et al.
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Article

Quantitative analysis of chromatin compaction in living cells using FLIM–FRET



David Llères1, John James2, Sam Swift2, David G. Norman3, and Angus I. Lamond1

1 Wellcome Trust Centre for Gene Regulation and Expression, 2 Microscopy Facility, and 3 Cancer Research UK Nucleic Acid Structure Research Group, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK

Correspondence to Angus I. Lamond: angus{at}lifesci.dundee.ac.uk

We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLaH2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.

Abbreviations used in this paper: 2-DG, 2-deoxyglucose; 5-FU, 5-fluorouracil; CENP-C, centromere protein C; FLIM, fluorescence lifetime imaging microscopy; FRET, Förster resonance energy transfer; H3K9triMe, histone H3 lysine trimethylation; MNase, micrococcal nuclease; MPLSM, multiphoton laser-scanning microscopy; PB, physiological buffer; ROI, region of interest; TCSPC, time-correlated single-photon counting; TEM, transmission EM; TSA, trichostatin A.

© 2009 Llères et al.
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