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

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

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Authors

  • David Lleres
  • John James
  • Sam Swift
  • David G. Norman
  • Angus I. Lamond (Lead / Corresponding author)

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Original languageEnglish
Pages481-496
Number of pages16
JournalJournal of Cell Biology
Journal publication date16 Nov 2009
Volume187
Issue4
DOIs
StatePublished

Abstract

We present a quantitative Forster 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.

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