Identification of nonhistone substrates of the lysine methyltransferase PRDM9.

Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone 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 nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone 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 nonhistone proteins. Using lysine-oriented peptide 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 nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.

赖氨酸甲基化是一种动态的翻译后标记，可调节组蛋白和非组蛋白蛋白质的功能。蛋白质。减数分裂重组期间的链破裂。确定PRDM9在任何组蛋白中都没有发现的甲基化肽序列然后，使用选择性曲线来识别假定的非组蛋白底物，这些底物通过胡椒斑阵列进行了测试，并通过体外KMT在重组蛋白上进一步验证了蛋白质水平的子集。用细胞中的PRDM9甲基化。