title: A sensitive, homogeneous, and high-throughput assay for lysine-specific histone demethylases at the H3K4 site.
abstract: Histone methylation is a regulated feature of nucleosomes that can have an impact on gene expression. The methylation state of histone residues 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 assay for histone demethylase activity at the H3K4 site has been developed in a time-resolved fluorescent resonance energy transfer assay format. The assay is based on the detection of the unmethylated H3 peptide by a fluorescent europium-chelate labeled monoclonal antibody binding specifically to the H3K4 site. The assay was validated for histone lysine-specific demethylase 1 and was demonstrated to be a suitable assay for inhibitor profiling and high-throughput screening.
authors.affiliation: Center for Proteomic Chemistry, Lead Finding Platform, Novartis Institute for Biomedical Research, Cambridge, Massachusetts 02139, USA.
authors.forename: Chunhua
authors.initials: C
authors.lastname: Wang
authors.forename: Mireille
authors.initials: M
authors.lastname: Caron
authors.forename: Debra
authors.initials: D
authors.lastname: Burdick
authors.forename: Zhaobin
authors.initials: Z
authors.lastname: Kang
authors.forename: Douglas
authors.initials: D
authors.lastname: Auld
authors.forename: William Adam
authors.initials: WA
authors.lastname: Hill
authors.forename: Jaime
authors.initials: J
authors.lastname: Padrós
authors.forename: Ji-Hu
authors.initials: JH
authors.lastname: Zhang
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D002614:Chelating Agents / Q000737:chemistry; D003627:Data Interpretation, Statistical; D005063:Europium; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D056466:Histone Demethylases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007202:Indicators and Reagents; D013379:Substrate Specificity
chemical_list: D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D056572:Histone Deacetylase Inhibitors; D007202:Indicators and Reagents; D005063:Europium; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D056466:Histone Demethylases; C495195:KDM1A protein, human