The MMSET protein is a histone methyltransferase specific for histones H3 and H4 in vitro. (A) The SET domain of MMSET catalyzes the transfer of ¹⁴C-methyl groups to histones H3 and H4. The GST fusion of the SET domain and the terminal PHD finger of MMSET was incubated with the indicated histone substrates and ¹⁴C-SAM. The reaction mix was resolved by SDS-PAGE, stained with Coomassie blue, whereas radiolabel retention was visualized by autoradiography. (B) The SET domains of MMSET and NSD1 catalyze the trimethylation of K20 in histone H4 in vitro. The methylation state of the recombinant histone was assayed by immunoblotting with antitrimethyl K20 antibodies. (C) The SET domains of MMSET and NSD1 catalyze histone H3 methylation in vitro. The methylation state of the recombinant histone was assayed by immunoblotting with corresponding antibodies. (D) A model structure of the SET domain of MMSET in complex with a substrate lysine as well as methyl-donor cofactor. Four residues in the MMSET active were predicted to play an important role in catalysis by mediating binding to the substrate histone, substrate lysine, and SAM cofactor. (E) Mutations of the identified residues greatly reduce HMT activity. Recombinant wild-type or mutant in each of the 4 identified SET domain residues was assayed for in vitro HMT activity with ¹⁴C-SAM.