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In vitro histone methyltransferase (HMT) assay
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The HMT reactions were performed by mixing purified 20 μg Dot1-TAP and 0.5 mg nucleosomes in the reaction buffer (50 mM Tris-HCl, pH 8.5, 50 mM NaCl, 5 mM MgCl2 and 1 mM DTT) with 10 mM SAM at 30 °C for 0–3 h. The reactions were quenched by adding 5×SDS buffer and incubated at 95 °C for 5 min. The products were resolved in a 10% or 18% SDS-PAGE gel and the histone methylation level was determined by immunoblotting with indicated antibodies.
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