In vitro histone demethylase assay.

ABSTRACT

Post-translational modifications of histones play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues, and methylation status defines the epigenetic program of a cell by determining chromatin structure and thereby regulating DNA-dependent processes such as transcription. Until recently, histone methylation was considered to be irreversible. However, the discovery of histone demethylases revealed that histone methylation is more dynamic than previously recognized. This protocol describes two different in vitro histone demethylase enzyme reactions and three different methods for measuring histone demethylase activity. The first reaction (type I) uses the Fe(II)- and α-ketoglutarate-dependent dioxygenase family of histone demethylase (represented by JmjC domain-containing histone demethylase [JHDM]); the second (type II) is for the flavin adenine dinucleotide (FAD)-dependent amine oxidase family (represented by lysine-specific demethylase 1 [LSD1]). Histone demethylase activity can then be detected by measuring the release of radiolabeled formaldehyde from (3)H-labeled methylated histone substrates, by monitoring the change in methylation levels of histone substrates by immunoblotting with site-specific methylhistone antibodies, or by using mass spectrometry to detect reductions in histone peptide masses that correspond to methyl groups. These assays can be applied to a wide range of histone demethylase studies, including the measurement of histone demethylase activity in tissue and cell lysates, identification of novel histone demethylases, and screening for inhibitors of histone demethylases.