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Automated, High Throughput, HTRF®-Based Detection of Histone Methyltransferase and Demethylase Enzyme Activity


Related Products: Synergy Neo2

January 14, 2013


Authors: Brad Larson and Peter Banks, BioTek Instruments, Inc., Winooski, Vermont, USA; Nicolas Pierre, Thomas Roux, Suzanne Graham and Francois Degorce, Cisbio US, Inc., Bedford, Massachusetts, USA

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The study of modifications which can affect the transcriptional state of DNA at the chromatin level, otherwise known as epigenetics, has seen increased emphasis in recent years. The alterations that have currently been described include, but are not limited to, acetyl- and deacetylation, methyl- and demethylation, ubiquitylation, and phosphorylation. These modifications take place mainly at the N-terminus of histone proteins, or histone tails, and affect gene expression in that portion of the DNA sequence. While epigenetic changes are normal, and essential, during the embryonic differentiation of cells from their original totipotent state, aberrant modifications have been linked to autoimmune disease, diabetes, and many human cancers.

Initial drug development in this area has concentrated on histone acetyltransferases (HATs) and histone deacetylases (HDACs). However, recent investigations have revealed that the process of histone methylation is also a dynamic process, controlled on one side mainly by the SET-domain protein methyltransferase family, and on the other by demethylases such as lysine-specifi c-demethylase-1 (LSD1) and JmjC domain-containing histone demethylase (JHDM). These two opposing processes are another important regulator of gene transcription. Abnormal histone methylation patterns, such as hypermethylation and hypomethylation, have been associated with human malignancies via multiple mechanisms including unscheduled gene silencing. As a result, the number of drug discovery projects focused on these two enzyme classes has increased in the last fi ve years and resulted in a number of promising new inhibitors currently in preclinical studies (Wagner, et al., 2012). Because of this recent trend, it has also become essential to have access to assay technologies that allow for easy assessment of new potential modulators of histone methylation in a high throughput format.

Here we describe two new HTRF®-based assay formats for the assessment of small molecule inhibitor capabilities of methylase and demethylase enzymes. The fi rst focuses on activity leading to monomethylation of histone H3 at the lysine 4 residue by the SET7/9 orphan member of the SET family. The second targets demethylation activity at the lysine 36 residue of histone H3 by the JMJD2A enzyme. Both assays are biochemical in nature and rely on the addition of a europium cryptate labeled antibody specifi c for the methylation state of the substrate, as well as a streptavidin-acceptor molecule, in a TR-FRET format. Enzyme activity in the well leads to the creation of the appropriate number of methyl groups on the substrate, allowing for antibody binding. Energy can then be transferred from donor to acceptor molecule, creating FRET. Inhibition of enzyme activity leads to the opposite effect, and decreasing FRET. The assay procedures were carried out using high throughput liquid handling and detection instrumentation. Optimization, validation, and screening data confi rm the ability of the automated process to deliver accurate results in a simple, yet robust manner.