Brad Larson, Peter Banks, Nicolas Pierre, Thomas Roux, Suzanne Graham, François Degorce
BioTek Instruments, Inc., Winooski, VT. Cisbio US, Bedford, MA, USA
Epigenetics is the study of modifications, which can affect the transcriptional state of DNA at the chromatin level. The epigenetics field has increased in recent years, and now includes research into alterations such as acetyl- and deacetylation, methyl- and demethylation, ubiquitylation, and phosphorylation. Modifications take place mainly at the histone protein N-terminus, or histone tail, and affect gene expression in that portion of the DNA sequence. These changes are a normal, essential part of a cell’s embryonic differentiation from its original totipotent state, however, aberrant modifications are linked to autoimmune disease, diabetes and many human cancers1. Epigenetic-based drug development has focused on histone acetyltransferases (HATs) and histone deacetylases (HDACs). However, recent research shows that histone methylation is also dynamic. It is controlled on one side mainly by the lysinespecific SET-domain protein methyltransferase family, and on the other side by demethylases such as lysine-specific demethylase 1 (LSD1) and JmjC domain-containing histone demethylase (JHDM). These opposing processes serve as another important gene transcription regulator. For example, abnormal histone methylation patterns, such as hyper- and hypomethylation, are associated with human malignancies via multiple mechanisms including unscheduled gene silencing. Many drug discovery projects now focus on methyltransferase and demethylase enzyme classes, and a number of promising new inhibitors are currently in preclinical studies2. Therefore, it is essential for assay technologies to allow easy assessment of new potential histone methylation modulators in high-throughput format. Here, we describe two homogeneous timeresolved fluorescence (HTRF®) assay formats from Cisbio Bioassays (Bedford, MA) to assess the small molecule inhibitor capabilities of methyltransferase and demethylase enzymes. The automated assay procedures were carried out using high-throughput liquid handling and detection instrumentation. Optimization, validation and screening data confirm that the automated process delivers accurate results in a simple yet robust manner.