Figure 6.

Specificity and affinity of binding to specific histone methyllysines was determined in a competitive binding assay. Unlabeled histone peptides displaced biotinylated histone H3 peptides, b-H3K4me3 and b-H3K9me3 (A), from methyl-binding domains. Unlabeled peptides were titrated into incubations containing 50 nM GST-MPP8 and 50 nM b-H3K9me3 (B and D), or 100 nM His-JMJD2A and 100 nM b-H3K4me3 (C and E). Varying incubation times of 15 min (triangles), 30 min (squares) and 60 min (circles) were examined for disruption of preformed biotinylated peptide/protein complex with unlabeled peptide (B and C). Displacement of b-H3K9me3 from GST-MPP8 and b-H3K4me3 from His-JMJD2A was measured following 30-min incubation with a range of unlabeled histone peptides (D and E, respectively). Data are expressed as the mean and standard deviation of four replicate measurements.