Stapled histone H3 tails are super-substrates for lysine methyltransferase SETD7.

The SETD7-catalysed methylation of lysine 4 in histone 3 (H3K4) plays an important role in the epigenetic control of eukaryotic gene expression. The N-terminal tail of histone H3 binds to SETD7 in a bend-like conformation in which Ala1 and Thr6 are located in close proximity, enabling the Lys4 substrate residue to react with the methyl group of the S-adenosylmethionine cosubstrate. Here, we report a proximity-guided design of H3 peptides stapled between amino acid residues 1 and 6 as potential substrates and inhibitors of human SETD7. Our results demonstrate that most of the appropriately stapled H3 peptides are efficiently methylated by SETD7, outperforming the unstructured, linear histone H3 tail sequence found in nature. The cyclic H3 peptides possessing the lactam linkage are excellent SETD7 substrates, outcompeting the linear H3K4 peptide, as demonstrated by up to 110-fold increase in catalytic efficiencies. The stapled H3 peptides display exclusive substrate selectivity for SETD7 over related H3K4 methyltransferases MLL3 and SETD1A. Inhibition assays show that the norleucine variant of the most efficient 1,6-stapled peptide substrate is a potent inhibitor of human SETD7. Overall, the results highlight a novel approach to selectively modulate the SETD7 activity and emphasise the potential of stapled histone peptides as exceptionally efficient peptidomimetic substrates and inhibitors of epigenetic enzymes.