A Sensitive, Homogeneous, and High-Throughput Assay for Lysine-Specific Histone Demethylases at the H3K4 Site

Abstract Histone methylationis a regulated feature of nucleosomesthat can have an impact on gene expression. The methylation state of histoneresidues has also been found in recent years to be associated with various disorders. Tools for detecting methylation state changes are very useful for dissecting the function of these epigenetic marks. In this work, a sensitive homogeneous assayfor histone demethylaseactivity at the H3K4 site has been developed in a time-resolved fluorescent resonance energy transfer assayformat. The assayis based on the detection of the unmethylated H3 peptide by a fluorescent europium-chelate labeled monoclonal antibody binding specifically to the H3K4 site. The assaywas validated for histonelysine-specific demethylase1 and was demonstrated to be a suitable assayfor inhibitor profiling and high-throughput screening.