Negative regulation of NF-κB action by Set9-mediated lysine methylation of the RelA subunit

Abstract

Proper regulation of NF‐κB activity is critical to maintain and balance the inflammatory response. Inactivation of the NF‐κB complex relies in part on the proteasome‐mediated degradation of promoter‐bound NF‐κB, but the detailed molecular mechanism initiating this process remains elusive. Here, we show that the methylation of the RelA subunit of NF‐κB has an important function in this process. Lysine methyltransferase Set9 physically associates with RelA in vitro and in vivo in response to TNF‐α stimulation. Mutational and mass spectrometric analyses reveal that RelA is monomethylated by Set9 at lysine residues 314 and 315 in vitro and in vivo. Methylation of RelA inhibits NF‐κB action by inducing the proteasome‐mediated degradation of promoter‐associated RelA. Depletion of Set9 by siRNA or mutation of the RelA methylation sites prolongs DNA binding of NF‐κB and enhances TNF‐α‐induced expression of NF‐κB target genes. Together, these findings unveil a novel mechanism by which methylation of RelA dictates the turnover of NF‐κB and controls the NF‐κB‐mediated inflammatory response.

Regulation of NF-κB activity through lysine monomethylation of p65

“…Two recent reports have also shown that NF-B is regulated by Set9 (19,24). In the first article, the authors show that Set9 regulates the expression of a subset of NF-B regulated genes in response to TNF␣ stimulation (19), which is consistent with our findings.…”

“…In the second study, the authors show that Set9 methylates p65 at K315 and K316 and negatively regulates the transcriptional activity of p65 (24). The differences in the in vitro data might result from different epitope tags used in generating the recombinant p65 fragments.…”

“…For example, the N-terminal GST tag used in the study might block the access of Set9 to K37 while GST-induced dimerization might facilitate the methylation of K315 and K316. The in vivo methylation results are more controversial as the antibody used in the study recognized dimethylated lysine according to the company Website (http:// www.abcam.com/pan-methyl-Lysine-methyl-K-pan-antibodyChIP-Grade-ab7315.html) whereas both K315 and K316 are monomethylated by Set9 (24). Furthermore, we tested the same antibody against p65 peptides (residues 33-41) with unmodified, mono-, di-or trimethylated K37 and found that the antibody recognized exclusively the dimethylated K37 p65 peptide (Fig.…”

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

“…Our study of NFκB methylation showed that the p65 subunit is not associated with histone-modifying enzymes until it is activated [7], suggesting that this event only happens after NFκB is released from IκB. Furthermore, the work of Yang et al [24] provided important evidence that the methylation of NFκB occurs only when it is in the nucleus and can bind to DNA, as a DNA binding-deficient mutant of p65 was no longer a substrate for methylation by SET7/9. Levy et al [26] showed that methylation of p65 by SETD6 occurs on the chromatin-associated protein.…”

“…Remarkably, the expression of the FBXL11 gene is induced in response to NFκB activation, forming a novel negative feedback loop similar to the one that involves the wellknown negative regulator IκB [23]. Yang et al [24] reported that K314 and K315 of p65 are monomethylated by SET7/9 in response to NFκB activation, an inhibitory modification that stimulates proteosome-mediated degradation of promoter-associated p65. Ea et al [25] reported that SET7/9 specifically methylates p65 at K37, and this methylation is restricted to the nucleus and regulates the promoter binding of p65.…”