In vitro histone demethylase assays

Abstract

Histone methylation plays pivotal roles in modulating chromatin structure and dynamics and in turn regulates genomic processes that require access to the DNA template. The methylation status at different sites is dynamically regulated by histone methyltransferases and demethylases. During the past decade, two classes of proteins have been characterized to actively remove methyl groups from lysine residues through different mechanisms. The LSD1/KDM1 family of amine oxidases require flavin adenine dinucleotide (FAD) for reaction, while the larger Jumonji C (JmjC) family of hydroxylases utilize Fe(II) and α-ketoglutarate as cofactors to demethylate histones. Since their discoveries, histone demethylases have been implicated in the precise control of gene expression program during development, cell identity, and fate decision. Several demethylases have also been linked to various human diseases such as neurological disorders and cancer. This chapter describes several in vitro assay conditions and detection methods for two classes of histone demethylases. We also discuss the protocols to prepare various substrates for different histone demethylase assays.