Posttranslational modification of histone tails including methylation, acetylation, or ubiquitination is an important epigenetic signal involved in gene and chromatin regulation (1,2). Histone lysine methyltransferases (HKMTs) are a large group of enzymes that specifically methylate histone tails at defined sites using S-adenosyl-L-methionine (AdoMet) as methyl group donor (3,4). Understanding the properties and mechanism of these enzymes is important, as many of them represent potential targets in cancer therapy (5). Different assays exist to determine the activity of HKMTs. Some use unlabeled AdoMet and detect the methylation of the peptide by mass spectrometry (6,7). Because of atomic mass resolution, mass spectrometric assays are very reliable and allow the distinction between mono-, di-, and trimethylated products (8). In addition, antibodies against different methylation states of lysines are used in the detection of histone methylation in fixed chromatin (9,10,11,12). Alternatively, the turnover of the coenzyme can be followed using a coupled fluorescent assay (13), in which the methyl donor product S-adenosy-L-homocysteine is enzymatically hydrolyzed to homocysteine and adenosine, and the homocysteine concentration is then determined by conjugation of its free sulfhydryl moiety to a fluorophore.

In an alternative approach, the transfer of a radioactively labeled methyl group from AdoMet to the peptide substrate can be detected. Radioactive assays need separation of modified peptide and unreacted AdoMet, which can be achieved by precipitation (14), gel electrophoresis (14,15,16), or using avidin/biotin technology if a biotinylated peptide is used as substrate (17). The biotin/avidin microplate peptide methylation assay previously described by us is convenient, very accurate, reproducible, and inexpensive (17). Since it yields quantitative results, it can be used for a characterization of the enzymatic properties of HKMTs and other protein methyltransferases. Also, the assay is well suited for high-throughput applications. However, one general disadvantage of all assays mentioned above is that they are discontinuous in nature.

Here, we have modified the setup of the radioactive avidin/biotin microplate assay to overcome this limitation ((Figure 1)A). To this end, avidincoated FlashPlate streptavidin 96-well scintillant-coated microplates (Perkin Elmer, Waltham, MA, USA) were used (18). In these plates, the interior of each well is permanently coated with a thin layer of polystyrene-based scintillant followed by covalent binding of streptavidin molecules. Using a biotin tag, the peptide is immobilized at the surface of the plate. After washing off the unbound peptide, the enzyme is added in buffer containing AdoMet bearing a tritiated methyl group. Due to the short range of the β-particles emitted by tritium, this does not lead to a strong scintillation signal, because most β-particles are quenched by solvent before they reach the wall of the plate. After enzymatic transfer of the radioactive methyl groups to the peptide substrates, however, they closely approach the walls of the microplate, which leads to a strong scintillation signal ((Figure 1)A).

Figure 1.

The Dim-5 histone 3 lysine 9 (H3K9) methyltransferase was expressed and purified as described (14). A synthetic peptide containing a biotin at its N terminus was purchased from IRIS Biotech (Marktredwitz, Germany) in high-performance liquid chromatography (HPLC)-purified form and was dissolved in water. Purity of the peptide was greater than 95%, as confirmed by HPLC and mass spectrometric analysis. The length of the peptide was 20 residues, corresponding to the first 19 amino acids of the histone H3 tail plus a methionine (Bt-MARTKQTARKSTGGKAPRKQ), with the target lysine residue bolded.