Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1

Nucleic Acids Research
Cecilia BergqvistEinar Hallberg

Abstract

In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that perip...Continue Reading

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Methods Mentioned

BETA
DamID
Fluorescence
fluorescence microscopy
PCR
transfection
electrophoresis
histone acetylation
biopsies
transfect

Software Mentioned

Excel
GraphPad Prism
ImageJ
ELYRA
Zen
PowerLogLogSlope
SnapGene
Zen Black 2011
CellProfiler

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