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Development of High-Throughput Assays to Study Methylases, Demethylases and Deacetylases Targeting Histone H3K4, H3K27 and H3K36 Residues
Mathieu Arcand, Mireille Caron, Julie Blouin, Claire Normand, Anne Labonté, Hendrick Plante, Lucille Beaudet & Jaime Padrós
PerkinElmer, 1744 William St., Montreal, QC H3J 1R4, Canada
Sponsored by PerkinElmer
Several assay methods have been developed for quantifying the activity of histone deacetylases (HDACs and sirtuins), histone methyltransferases (HMTs), and histone demethylases (HDMs). These include radioactive assays, enzymelinked immunoassays (ELISA), mass spectrometry, and enzyme-coupled detection of fluorescent peptides or reaction co-products (e.g. S-adenosylhomocysteine, formaldehyde, hydrogen peroxide). These assays suffer from various drawbacks such as low throughput, lack of sensitivity, generation of hazardous waste, requirement for expensive equipment, or artifacts associated with the use of nonphysiological fluorescent moieties or enzyme-coupled assays (generation of false positives/negatives).
In this study, we describe the development and optimization of homogeneous antibody-based assays for measuring the catalytic activity of a series of epigenetic lysine-modifying enzymes acting on histone H3 Lys4 (SIRT1 deacetylase and LSD1 demethylase), Lys27 (HDAC1 deacetylase, EZH2 methyltransferase and JMJD3 demethylase) and Lys36 (JMJD2A demethylase). Two different non-radioactive, no-wash technologies were used for detection of the enzymatic reaction products: amplified luminescent proximity homogeneous (AlphaLISA®) assay and time-resolved Förster energy transfer (LANCE® Ultra) assay.
Results demonstrated that all assays were sensitive, rapid and robust (Z’ factors ≥ 0.69), requiring only nanomolar concentrations of enzyme and peptide. Furthermore, profiling of known inhibitors for each epigenetic enzyme showed the expected potency with either technology. These assays will therefore be ideal for the identification of selective small molecule inhibitors. The approach described here is broadly suitable for measuring the catalytic activity of other histonemodifying enzymes by combining the appropriate biotinylated histone-derived peptides and mark-selective antibodies.
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