Abstract
Post-translational histone modifications are major regulators of gene expression. However, conventional immunoassays do not provide sufficient information regarding their spatial and temporal dynamic changes. Fluorescence/Förster resonance energy transfer (FRET)-based probes are capable of monitoring the dynamic changes associated with histone modifications in real-time by measuring the balance between histone-modifying enzyme activities. Recently, a genetically encoded histone-modification fluorescent probe using a single-chain variable region (scFv) fragment of a specific antibody was developed. The probe, modification-specific intracellular antibody, is capable of monitoring histone-acetylation levels in both cultured cells and living organisms based on the ratio of fluorescence intensities between the cell nucleus and cytoplasm. In this study, we constructed a FRET probe composed of yellow fluorescent protein attached at the N-terminus of an acetyl H3K9-specific scFv, tethered to a cyan fluorescent protein. When the FRET probe was expressed in human cells, both FRET efficiency and fluorescence intensity in the nucleus increased following histone-deacetylase inhibitor treatment. Using these two parameters, endogenous histone-acetylation levels were quantified over a high dynamic range. This probe provides a simple approach to quantify spatial and temporal dynamic changes in histone acetylation.
Highlights
Histones are relatively small proteins that associate with and help package DNA into chromatin in the nucleus
The dynamic changes in the fluorescence resonance energy transfer (FRET) efficiency of intracellular probes are monitored as the fluorescence-intensity ratio of the acceptor to the donor
We developed a single-chain fusion protein consisting of two differently colored fluorescent proteins and an intrabody that associates with acetylated histone H3K9 as a probe[17]
Summary
Histones are relatively small proteins that associate with and help package DNA into chromatin in the nucleus. A longer-lived genetically encoded fluorescent probe, modification-specific intracellular antibody (mintbody), was developed[14] This probe was composed of a single-chain variable-region (scFv) fragment capable of being functionally expressed in the reductive cellular environment (intrabody) and tethered to an enhanced green fluorescent protein (EGFP) fused to its C-terminus. This probe retained high specificity for H3K9 acetylation and was successful in monitoring histone-acetylation levels in cultured cells and living organisms by tracking the nuclear:cytoplasmic intensity ratio of EGFP. After challenge with histone-deacetylase inhibitor, both FRET efficiency and nuclear-fluorescence intensity increased in a time-dependent manner Using these two parameters, endogenous histone-acetylation levels were capable of being quantified with a high dynamic range
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