The SET8 H4K20 protein lysine methyltransferase has a long recognition sequence covering seven amino acid residues.

The SET8 histone lysine methyltransferase, which monomethylates the histone 4 lysine 20 residue plays important roles in cell cycle control and genomic stability. By employing peptide arrays we have shown that it has a long recognition sequence motif covering seven amino acid residues, viz. R(17)-H(18)-(R(19)KY)-K(20)-(V(21)ILFY)-(L(22)FY)-R(23). Celluspots peptide array methylation studies confirmed specific monomethylation of H4K20 and revealed that the symmetric and asymmetric methylation on R(17) of the H4 tail inhibits methylation on H4K20. Similarly, dimethylation of the R located at the -3 position also reduced methylation of p53 K382 which had been shown previously to be methylated by SET8. Based on the derived specificity profile, we identified 4 potential non-histone substrate proteins. After relaxing the specificity profile, we identified several more candidate substrates and showed efficient methylation of 20 novel non-histone peptides by SET8. However, apart from H4 and p53 none of the identified novel peptide targets was methylated at the protein level. Since H4 and p53 both contain the target lysine in an unstructured part of the protein, we conclude that the long recognition sequence of SET8 makes it difficult to methylate a lysine in a folded region of a protein, because amino acid side chains essential for recognition will be buried.