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Sci. Signal., 17 November 2009
Vol. 2, Issue 97, p. ec371
[DOI: 10.1126/scisignal.297ec371]

EDITORS' CHOICE

Posttranslational Modifications Picking a Promoter

Nancy R. Gough

Science Signaling, AAAS, Washington, DC 20005, USA

The transcription factor NF-{kappa}B plays key roles in inflammatory and immune responses. In different contexts, NF-{kappa}B regulates different sets of genes. Previous work had suggested that the methyltransferase Set9 influenced the expression of NF-{kappa}B–responsive genes. Ea and Baltimore show that treatment of human embryonic kidney (HEK) 293 cells with a nonspecific methyltransferase inhibitor decreased activation of an NF-{kappa}B reporter gene in response to tumor necrosis factor–{alpha} (TNF-{alpha}). When recombinant Set9 was incubated in vitro with peptides from the p65 subunit of NF-{kappa}B, peptides containing Lys37 were methylated. To investigate the function of NF-{kappa}B Lys37 methylation in vivo, they developed an antibody specific for this modified form of NF-{kappa}B and verified its specificity with a mutant (K37Q) that could not be methylated. In Western blot experiments, the antibody detected methylated NF-{kappa}B in the nuclear fractions, but not the cytosol, of cells treated with TNF-{alpha} or interleukin-1β (IL-1β). Lys37 methylation in response to TNF-{alpha} or IL-1β was undetectable in cells in which Set9 was knocked down with short-interfering RNAs. Selected induction of specific NF-{kappa}B–responsive genes was inhibited in cells in which Set9 was knocked down: Induction of the genes encoding TNF-{alpha} and the chemokine CXCL10 was decreased, but induction of the gene encoding I{kappa}B{alpha} was not. Electrophoretic mobility shift assays performed under high salt conditions on nuclear extracts from control or Set9-knockdown cells suggested that Lys37 methylation stabilized a subset of p65-DNA interactions, a finding confirmed by chromatin immunoprecipitation experiments. Induction of the genes encoding TNF-{alpha} and CXCL10 was only fully restored by reconstitution of p65-deficient mouse embryo fibroblasts with wild-type p65 and not the K37Q mutant. The authors suggest that this modification may be context-specific and speculate that lysine methylation of this N-terminal lysine (Lys37) may be important for gene activation, whereas lysine methylation of more C-terminal residues (Lys315, Lys316) may be important for termination of activity, a phenomenon that had been reported previously.

C.-K. Ea, D. Baltimore, Regulation of NF-{kappa}B activity through lysine monomethylation of p65. Proc. Natl. Acad. Sci. U.S.A. 106, 18972–18977 (2009). [Abstract] [Full Text]

Citation: N. R. Gough, Picking a Promoter. Sci. Signal. 2, ec371 (2009).



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