Monomethylation of histone 3 lysine 9 (H3K9me1) is a transcriptional repressive mark of importance during embryonical development and involved in cancerous and autoimmune diseases. Finding substances that interfere with methyltransferases that add the methyl group onto the histone is accordingly important.
The assay presented here uses unmethylated but biotin-labelled H3 peptides as a substrate. Addition of the histone methyltransferase G9a and the cofactor S-adenosylmethionine results in H3K9me1. The methylated peptide can be bound by an HTRF® donor antibody whereas a streptavidin-coupled acceptor binds each H3 peptide. Upon excitement of the Eu3+ cryptate donor its emsission may in turn excite the acceptor if bound on the same peptide.
The assay allowed determination of the Km value of the substrate and determination of the IC50 values of histone methyltransferase inhibitors.
Reading the HTRF assay on a PHERAstar® provided a rapid analysis of histone methyltransferase activity in 384 well plates.