G9a-mediated Lysine Methylation Alters the Function of CCAAT/Enhancer-binding Protein-β

Abstract

The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-␤ (C/EBP␤) 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/EBP␤ transactivation domain (TAD). Binding between G9a and C/EBP␤ was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBP␤ is post-translationally modified by methylation in vivo. A conserved lysine residue in the C/EBP␤ 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/EBP␤. A C/EBP␤ TAD mutant that contained a lysine-toalanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBP␤ target gene. Our data identify C/EBP␤ as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBP␤ during gene regulation.

The E3 Ubiquitin Ligase Neuregulin Receptor Degradation Protein 1 (Nrdp1) Promotes M2 Macrophage Polarization by Ubiquitinating and Activating Transcription Factor CCAAT/Enhancer-binding Protein β (C/EBPβ)

“…C/EBP␤ transcription efficacy can be regulated by lots of post-translational modifications, including phosphorylation (31,32), SUMOylation (33), acetylation (34), and methylation (35). In addition, it has been shown that phosphorylation of C/EBP␤ can abrogate its methylation by protein-arginine methyltransferase 4 PRMT4/CARM1, thus demonstrating the cross-talk between phosphorylation and epigenetic methylation of C/EBP␤ (36).…”

Interaction and Cooperation of the CCAAT-box Enhancer-binding Protein β (C/EBPβ) with the Homeodomain-interacting Protein Kinase 2 (Hipk2)

“…Recent work has shown that phosphorylation of Thr-220 triggers a cascade of events involving abrogation of the binding of the lysine methylase G9a and arginine methyl transferase PRMT4, thereby altering the pattern of covalent post-translational modifications, in particular the PRMT4-dependent methylation of arginine 3 of C/EBP␤. This, in turn, facilitates the recruitment of SWI/SNF chromatin remodeling and Mediator complexes by C/EBP␤ to ultimately increase the transcription of C/EBP␤ target genes (26,27,49). Our work suggests increased binding and phosphorylation of p300 as an alternative or additional mechanism by which the phosphorylation of C/EBP␤ at Thr-220 stimulates the transactivation potential of C/EBP␤.…”

“…The relative amounts of the different isoforms vary between different cell types and are influenced via a short upstream open reading frame by the status of the cell (11,(13)(14)(15). In addition to differential isoform expression, C/EBP␤, is regulated by posttranslational modifications, such as phosphorylation (16 -18), acetylation (19 -21), and sumoylation (22) as well as by the binding of specific cofactors via protein-protein interactions (23)(24)(25)(26)(27). Besides its function in normal cells, deregulation of C/EBP␤ activity or isoform expression contributes to the development of cancer (28 -31).…”

Lysine methylation of promoter-bound transcription factors and relevance to cancer

“…Several nonhistone proteins can be methylated by G9a. The CCAAT/enhancer-binding protein-β (C/EBPβ) is methylated at K39, which may create a binding site for a repressive protein complex or enhance interaction with C/EBPβ by "reading" methylated K39 [12]. Lee et al [37] reported that reptin, a chromatin-remodeling factor, is methylated at K67 under hypoxic conditions by G9a.…”

“…Moreover, increasing evidence indicates that nonhistone proteins are subject to reversible acetylation or methylation by histone-modifying enzymes. Among these nonhistone targets are transcription factors, hormone receptors, signal transducers, chaperones, and proteins of the cytoskeleton [6][7][8][9][10][11][12]. Although the acetylation of nonhistone proteins has been appreciated for some time [9,13,14], their methylation has been recognized only more recently.…”