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

TY - JOUR

T1 - Quantitative analysis of chromatin compaction in living cells using FLIM-FRET

A1 - Lleres,David

A1 - James,John

A1 - Swift,Sam

A1 - Norman,David G.

A1 - Lamond,Angus I.

AU - Lleres,David

AU - James,John

AU - Swift,Sam

AU - Norman,David G.

AU - Lamond,Angus I.

PY - 2009/11/16

Y1 - 2009/11/16

N2 - <p>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.</p>

AB - <p>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.</p>

KW - LIFETIME IMAGING MICROSCOPY

KW - HISTONE ACETYLATION

KW - IN-VIVO

KW - FLUORESCENT PROTEINS

KW - TIME-LAPSE

KW - HETEROCHROMATIN

KW - DYNAMICS

KW - NUCLEOSOME

KW - TRANSCRIPTION

KW - FIBER

U2 - 10.1083/jcb.200907029

DO - 10.1083/jcb.200907029

M1 - Article

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 4

VL - 187

SP - 481

EP - 496

ER -