Specificity of the SUV4–20H1 and SUV4–20H2 protein lysine methyltransferases and methylation of novel substrates

• SUV4–20H1 and H2 strongly prefer H4K20me1 substrates and introduce one methyl group
• Both recognize the + 1, + 2 and + 4 sites of peptide substrates, SUV4–20H1 also − 1.
• We describe novel non-histone substrates of SUV4–20H1 and H2
• In vitro methylation of ERK1 by SUV4–20H1 or H2 was not observed

The SUV4–20H1 and SUV4–20H2 enzymes methylate histone H4 at K20, and they have overlapping and distinct biological effects. Here, by in vitro methylation studies we confirmed that both the murine SUV4–20H enzymes strongly favor the monomethylated H4K20 peptide substrate. We also show that both enzymes only generate dimethylated H4K20 products. We determined the substrate sequence recognition motif of both enzymes using SPOT peptide arrays showing that SUV4–20H1 recognizes an (RY)-Kme1-(IVLM)-(LFI)-X-D sequence. In contrast, SUV4–20H2 shows less specificity and recognizes an X-Kme1-(IVLMK)-(LVFI)-X-(DEV) sequence, which is partially overlapping with SUV4–20H1 but has relaxed specificity at the − 1 and + 4 positions (if the target H4K20me1 is positon 0). Based on our data, we identify novel peptide substrates for SUV4–20H1 (K1423 of Zinc finger protein castor homolog 1) and SUV4–20H2 (K1423 of Zinc finger protein castor homolog 1, K215 of Protein Mis18-beta and K308 of Centromere protein U). All these lysine residues were already identified to be methylated in human cells, but the responsible PKMT was not known. In addition, we also tested the activity of SUV4–20H enzymes on ERK1, which was recently reported to be methylated by SUV4–20H1 at K302 and K361. However the sequences surrounding both methylation sites do not fit to the specificity profile of SUV4–20H1 and we could not detect methylation of ERK1 by any of the SUV4–20H enzymes. The possible reasons of this discrepancy and its consequences are discussed.

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