G9a and Glp1 methylate H1.2K187. (a) Amino acid sequence of the C-terminal tail of H1.2. Lysines as potential methylation sites are highlighted in grey. (b) Cluster mutation approach to map methylation sites in the C-terminal part with its 40 potential sites. Cluster definitions and their substituting amino acid sequence are depicted. All lysines in the C-terminal tail of H1.2 were classified into 10 clusters (grey rectangles). Cluster 12 is a fusion of clusters 1 and 2. (Upper) Wild-type H1.2 sequence; (lower) the substituting amino acids. (c) The methylated site is between amino acids 187 and 196. Methylation assay of the cluster mutation constructs with immunoprecipitated Glp1. Used constructs are schematically indicated on top. Grey rectangles, mutated clusters; white rectangles, wild-type clusters. In addition, all constructs carry a lysine to arginine mutation introducing an arginase C cutting site. (Upper panel) Ponceau (Pon) staining as a loading control. The different migration levels of the constructs are due to the substitutions and replacements of different numbers of uncharged and positively charged lysines by uncharged amino acids. Note a strong reduction in methylation in lane 16, corresponding to a cluster 8 mutation (mutated cluster containing K187 is indicated by asterix). (d) Methylation assay with wild-type H1.2CT, H1.2CT K172R, H1.2CT K187R, H1.2CT K172R and K187R and wild-type H1.2CT (2× z-tag) as substrates. Mutation of K187 results in a strong reduction of methylation by immunoprecipitated Glp1. (Upper panel) Pon; (lower panel) autoradiography (Rad). Wild-type H1.2CT (2× z-tag) was added to the methylation assays as an internal control of methyltransferase activity (lanes 5 to 8). (e) Methylation assays on peptides. Peptides containing unmodified K187, monomethylated K187 and dimethylated K187 and immunoprecipitated G9a were used for methylation assay. Rad is shown. Note that unmodified and monomethylated peptide were methylated to similar extents, whereas the dimethylated peptide was not methylated.