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Assays for Validating Histone Acetyltransferase Inhibitors
Published on: August 6, 2020
In vitro histone demethylase assay.
Yu-ichi Tsukada1, Keiichi I Nakayama
1Division of Cell Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
Cold Spring Harbor Protocols
|October 5, 2010
Summary
Histone demethylases dynamically regulate gene expression by removing methyl groups from histones. This study details in vitro enzyme reactions and assays to measure histone demethylase activity, crucial for epigenetic research.
Area of Science:
- Epigenetics and Molecular Biology
- Chromatin Biology
- Enzymology
Background:
- Histone methylation is a key epigenetic mark regulating chromatin structure and gene transcription.
- Previously considered irreversible, histone methylation is now understood to be dynamic due to histone demethylases.
- Histone demethylases are enzymes that remove methyl groups from histone proteins, influencing cellular epigenetic programs.
Purpose of the Study:
- To describe protocols for in vitro histone demethylase enzyme reactions.
- To present methods for measuring histone demethylase activity.
- To facilitate the study of histone demethylases in various biological contexts.
Main Methods:
- Describes two types of in vitro histone demethylase reactions: Fe(II)- and α-ketoglutarate-dependent dioxygenase (e.g., JHDM) and FAD-dependent amine oxidase (e.g., LSD1).
- Details three methods for detecting histone demethylase activity: radiolabeled formaldehyde release, immunoblotting with methylhistone antibodies, and mass spectrometry.
- These assays enable the quantification of enzyme activity using various histone substrates.
Main Results:
- Established protocols for assaying histone demethylase activity.
- Demonstrated the utility of different detection methods for measuring enzyme function.
- Provided a framework for studying histone demethylase kinetics and inhibition.
Conclusions:
- Histone methylation dynamics are regulated by demethylase enzymes.
- The described in vitro assays are versatile for studying histone demethylases.
- These methods support research in epigenetics, including inhibitor screening and novel demethylase discovery.
