Abstract 64: Development of homogeneous non-radioactive assays for studying histone 3 methyltransferases and demethylases

Abstract

Abstract Post-translational modifications of histones are among the epigenetic mechanisms that can affect chromatin structure and function. Disruption of epigenetic processes can lead to altered gene expression and malignant cellular transformation. Epigenetic changes including histone acetylation, histone methylation and DNA methylation are now considered to play important roles in the initiation and progression of cancer. Histone methylation and demethylation are enzymatically dynamic processes controlled respectively by histone methyltransferases (HMTs) and histone demethylases (HDMs). Several assay methods have been developed for quantifying the activity of HMTs and HDMs. These include radioactive assays, enzyme-linked immunoassays (ELISA), mass spectrometry, and enzyme-coupled detection of 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 enzyme-coupled assays (generation of false positives/negatives). In this study, we describe homogeneous (mix-and-read) assays for measuring the catalytic activity of both HMTs and HDMs acting on histone H3 using two different non-radioactive technologies: amplified luminescent proximity homogeneous assay (AlphaLISA) and time-resolved fluorescence energy transfer (LANCE). The EZH2 complex (which methylates histone H3 on lysine 27) and LSD1 and JMJD2A (which demethylate di-methyl lysine 4 and tri-methyl lysine 36, respectively, on histone H3) were selected as model enzymes due to their association with various human diseases. Assays were developed in 384-well format and used as substrates two synthetic biotinylated peptides derived from the N-terminus of histone H3 (amino acids 1 to 21 for LSD1 and 21 to 44 for EZH2 and JMJD2A). All assays were designed as signal-increase homogeneous assays, where direct detection of product formation was conducted using methyl-state selective antibodies conjugated to either AlphaLISA acceptor beads or LANCE europium chelate. Results demonstrated that all assays were sensitive and robust. 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.Although these studies focused on EZH2, JMJD2A and LSD1 enzymes, the approach described here is broadly suitable for measuring the catalytic activity of other HMTs and HDMTs by combining the appropriate biotinylated histone-derived peptides and methyl-state selective antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 64. doi:10.1158/1538-7445.AM2011-64