Identification of non-histone substrates of the lysine methyltransferase PRDM9.

Lysine methylation is a dynamic, post-translational mark that regulates the function of histone and non-histone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate non-histone proteins. In this work, we investigate the substrate selectivity of the lysine methyltransferase PRDM9 to identify both potential histone and non-histone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on non-histone proteins. Using lysine-oriented peptide (K-OPL) libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative non-histone substrates, which were tested by peptide spot array, and a subset were further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the non-histone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.