Intrabody-based FRET probe to visualize endogenous histone acetylation

Area of Science:

• Molecular Biology
• Epigenetics
• Biochemistry

Background:

• Post-translational histone modifications regulate gene expression but are difficult to track dynamically.
• Conventional immunoassays lack spatial and temporal resolution for these modifications.
• Fluorescence/Förster resonance energy transfer (FRET)-based probes offer real-time monitoring of histone modification dynamics.

Purpose of the Study:

• To develop a genetically encoded FRET probe for quantifying dynamic changes in histone acetylation.
• To assess the probe's ability to monitor histone acetylation levels in human cells and living organisms.
• To establish a simple approach for high dynamic range quantification of spatial and temporal histone acetylation changes.

Main Methods:

• Construction of a FRET probe using yellow fluorescent protein, an acetyl H3K9-specific single-chain variable region (scFv) fragment, and cyan fluorescent protein.
• Expression of the FRET probe in human cells.
• Treatment of cells with histone-deacetylase inhibitors.
• Measurement of FRET efficiency and nuclear fluorescence intensity.

Main Results:

• The FRET probe successfully monitored histone acetylation levels in human cells.
• Both FRET efficiency and nuclear fluorescence intensity increased upon histone-deacetylase inhibitor treatment.
• Endogenous histone acetylation levels were quantified over a high dynamic range using the probe's parameters.

Conclusions:

• The developed FRET probe provides a simple and effective method for quantifying spatial and temporal dynamic changes in histone acetylation.
• This tool advances the study of gene expression regulation by enabling real-time monitoring of epigenetic modifications.
• The probe has potential applications in both cultured cells and living organisms for epigenetic research.