Histone Methyltransferase Activity Assay

The HMT assay was performed as described in Iglesias et al., 2018 (link). Briefly, the [3H]-based HMT assay was carried out in a 10 µl reaction mix containing 2 µM histone or nucleosome, 5.6 µM [3H]- S-adenosyl methionine (SAM), 0.3–1 µM enzyme, and 1x HMT buffer (50 mM Tris-HCl, pH 8.0, 20 mM KCl, 10 mM MgCl₂, 0.02% Triton X-100, 1 mM DTT, 5% glycerol, and 1 mM PMSF). The reaction mix was incubated at 20°C for 2 hr, and then loaded onto 17% SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membrane, which was then soaked with autoradiography enhancer (EN3HANCE, PerkinElmer) and then air dried. Fluorography signal was detected by X-ray film. For the histone mass spectrometry analysis, 150 µl reaction mix containing approximately 0.3 µM GST-SET-32, 2.5 µM H3, and 213 µM SAM and 1 x HMT buffer without Triton X-100 was used.