G9a-mediated lysine methylation alters the function of CCAAT/enhancer-binding protein-beta

The functional capacity of the transcriptional regulatory CCAAT/Enhancer Binding Protein (C/EBP) ss is governed by protein interactions and post-translational protein modifications. In a proteome wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine-9 specific 3 (G9a) was found to directly interact with the C/EBPss transactivation domain (TAD). Binding between G9a and C/EBPss was confirmed by GST-pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBPss is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBPss-TAD served as a substrate for G9a mediated methylation. G9a, but not a methyltransferase defective G9a mutant, abrogated the transactivation potential of wild type C/EBPss. A C/EBPss TAD mutant that contained a lysine to alanine exchange was resistant to G9a mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin embedded, endogenous C/EBPss target gene. Our data identify C/EBPss as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBPss during gene regulation.