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 and Jaime Padrós Bio-Discovery, PerkinElmer, 1744 William, Suite 600, Montreal, Quebec, Canada H3J 1R4
Post-translational modifications (PTMs) of histones are among the epigenetic mechanisms that can affect chromatin structure and function. Although histones are subject to a myriad of PTMs including phosphorylation, ubiquitination, and glycosylation on various residues, there has been a focus on histone-modifying enzymes catalyzing reversible lysine acetylation and methylation. Indeed, these lysine-modifying enzymes have been associated with several diseases, making them potential targets for epigenetic therapy. 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, enzyme-linked 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 non-physiological 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®) assay. Assays were developed in 384-well format using as substrates two synthetic biotinylated peptides derived from the N-terminus of histone H3 (amino acids 1 to 21 for LSD1 and SIRT1 assays, and 21 to 44 for EZH2, JMJD2A and JMJD3 assays). Except for HDAC1, all assays were designed as signal-increase homogeneous assays. In all cases, direct detection of product formation was conducted using modification-specific antibodies conjugated to either AlphaLISA acceptor beads or LANCE europium chelate. Results demonstrated that all assays were sensitive, rapid and robust (Z’ factors greater than 0.7), requiring only nanomolar concentrations of enzyme and peptide and exhibiting low substrate turnover (<20%). 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 histone-modifying enzymes by combining the appropriate biotinylated histone-derived peptides and mark-selective antibodies.
