Regulation of NF-kappaB by NSD1/FBXL11-dependent reversible lysine methylation of p65

Abstract

NF-kappaB, a central coordinator of immune and inflammatory responses, must be tightly regulated. We describe a NF-kappaB regulatory pathway that is driven by reversible lysine methylation of the p65 subunit, carried out by a lysine methylase, the nuclear receptor-binding SET domain-containing protein 1 (NSD1), and a lysine demethylase, F-box and leucine-rich repeat protein 11 (FBXL11). Overexpression of FBXL11 inhibits NF-kappaB activity, and a high level of NSD1 activates NF-kappaB and reverses the inhibitory effect of FBXL11, whereas reduced expression of NSD1 decreases NF-kappaB activation. The targets are K218 and K221 of p65, which are methylated in cells with activated NF-kappaB. Overexpression of FBXL11 slowed the growth of HT29 cancer cells, whereas shRNA-mediated knockdown had the opposite effect, and these phenotypes were dependent on K218/K221 methylation. In mouse embryo fibroblasts, the activation of most p65-dependent genes relied on K218/K221 methylation. Importantly, expression of the FBXL11 gene is driven by NF-kappaB, revealing a negative regulatory feedback loop. We conclude that reversible lysine methylation of NF-kappaB is an important element in the complex regulation of this key transcription factor.

Fig. 1.

NSD1 activates NF-κB and reverses the inhibitory effect of FBXL11; FBXL11 and NSD1 bind to p65 upon NF-κB activation. (A) Luciferase assay of NF-κB activity. Expression of NSD1 activates NF-κB and reverses inhibition caused by increased expression of FBXL11 in different cells, including 293C6, HT29, and mutant Z3 cells, and corresponding cells in which FBXL11 is overexpressed. Results of triplicate luciferase assays are shown as means ± SD. (B) (Left) Western assay showing knockdown of expression of NSD1 in 293C6 cells with shRNA. (Right) Luciferase assay of NF-κB activity in 293C6 cells treated with IL-1β for different times, showing that reduced expression of NSD1 by shRNA decreased NF-κB activity. Results of triplicate luciferase assays are shown as means ± SD. (C) EMSA assay of NF-κB in 293C6 cells treated with IL-1β for different times, showing that reduced NSD1 expression by shRNA decreased NF-κB DNA binding activity. (D and E) p65 Coimmunoprecipitates with FBXL11 and NSD1; FBXL11 and NSD1 bind to p65 only when NF-κB is activated. Immunoprecipitation (IP) assays detect binding of endogenous FBXL11 or NSD1 to p65, either in 293C6 cells treated with IL-1β (Upper) or in HT29 cells with constitutive NF-κB activation (Lower).

Fig. 2.

Mass spectrometry (MS) shows that p65 is methylated on K218 and K221 on NF-κB activation. (A) GelCode blue-stained gel, showing that p65 is immunoprecipitated as a single strong band. The same samples were loaded into multiple lanes. (B) Analysis of tryptic peptides derived from p65 suggests that K218 is monomethylated. The pure single band was digested in the gel and samples were analyzed by LC-MS/MS. A mass shift of +14 was observed spanning peptide 202–218. Tandem MS analysis further suggested that K218 on the C-terminal side of the peptide is modified. Another +14 mass shift was also observed on the N-terminal side, suggesting an additional methylation within the N-terminal four residues. (C) Analysis of tryptic peptides derived from p65 suggests that K221 of p65 is dimethylated. A mass shift of +28 was observed spanning peptide 219–236. Tandem MS analysis further suggested that K221 on the N-terminal is dimethylated.

Fig. 3.

K218 and K221 are functional sites that are methylated on constitutive activation of NF-κB and demethylated on expression of FBXL11. (A) EMSA assay, showing that p65 mutant proteins K218A, K221A, and K218/221A differentially inhibited the ability of NF-κB to bind to DNA in 293C6 cells. (Lower) Western analysis showing that expression levels of the different p65 constructs are similar. (B) Mutations of K218, K221, and K218/221 of p65 differentially decreased NF-κB activity and the inhibitory effect of FBXL11 on NF-κB in 293C6 cells. (Left) K-A mutants. (Right) K-R mutants. Luciferase assay shows level of NF-κB activity after each mutant was transiently expressed for 48 h. Results of triplicate luciferase assays are shown as means ± SD. (C) Mutations K218A, K221A, and K218/221A of p65 differentially decreased expression of the typical NF-κB target gene E-SELECTIN and the inhibitory effect of FBXL11 on NF-κB in 293C6 cells (Left) and HT29 cells (Right). The different p65 constructs were transfected transiently into 293C6 or HT29 cells and, 48 h later, samples were collected for Northern analyses. (D) Western analyses, showing that p65 is monomethylated on K218 and dimethylated on K221 on constitutive activation of NF-κB, driven by the overexpression of wild-type p65, but not the K218/K221A mutant protein. The degree of methylation was dramatically decreased by the expression of FBXL11 in both 293C6 (Left) and HT29 cells (Right).

Fig. 4.

K218 and K221 residues of p65 are methylated and demethylated by the NSD1-FBXL11 enzyme pair in response to cytokines; K218 and K221 are methylated in response to dsRNA or LPS. (A) Western analysis, showing that K218 and K221 of p65 are methylated and demethylated by NSD1 and FBXL11, respectively, in response to cytokines. 293C6 (Left) or HT29 cells (Right), and the corresponding FBXL11 or NSD1 cells, were treated with IL-1β or TNFα for 45 min before being collected for Western analyses. (B) K218 and K221 of p65 are methylated in response to either dsRNA in 293C6-TLR3 cells (Left) or LPS in 293C6-TLR4 cells (Right).

Fig. 5.

Effects of FBXL11 expression on cell proliferation and colony formation. (A) K218/221-dependent effect of FBXL11 on cell proliferation in colon cancer HT29 cells. High levels of FBXL11 decrease cell growth, whereas decreasing FBXL11 expression with shRNA increases cell growth in vector-transfected cells. However, knocking down p65 expression with an shRNA against its 5′-UTR greatly decreased the proliferation of HT29 cells and of cells expressing FBXL11 or shRNAs to FBXL11. The phenotype caused by shRNA to p65 can be reversed by expression of wild-type p65 but not the K218/221A mutant. (B) K218/221-dependent effect of FBXL11 on colony formation. (Upper) Images of colonies in a soft agar assay, showing that high levels of FBXL11 decrease sizes of colonies formed, whereas decreased expression of FBXL11 increases sizes of colonies in vector-transfected cells. However, knocking down p65 expression with shRNA against its 5′-UTR greatly decreases size of colonies formed by HT29 cells and cells expressing FBXL11 or shRNA to FBXL11. Phenotype caused by shRNA to p65 can be reversed by expression of wild-type p65 but not the K218/221A mutant protein. (Lower) Statistical analysis of the sizes of the colonies observed. Results of triplicate assays are shown as means ± SD.

Fig. 6.

Regulation of p65-dependent gene expression by FBXL11 in MEFs. (A) Correlation between wild-type (WT) p65, K218/221A (KA) mutant p65, and FBXL11 on p65-dependent gene expression in p65-null MEFs. In the WT ± FBXL11 sample, “Dependent” represents genes that are downregulated by FBXL11. In the WT vs. 218/221 double KA sample, “Dependent” represents genes that are p65 dependent but not activated after expression of the KA mutant protein. In the KA ± FBXL11 sample, “Dependent” represents KA-affected genes that are also regulated by FBXL11. Constructs encoding wild-type or double KA mutant proteins were transfected transiently into p65-null MEFs and, 48 h later, samples were collected and RNAs were purified for analysis. Expression of wild-type p65 caused constitutive activation of NF-κB, leading to the induction of gene expression, which is designated as p65 dependent. The fold increase cutoff for analysis is 2. (B) Confirmation of the expression of some genes from the array analysis in MEFs. The same RNA samples were used for the Northern experiment. Probes for IGF1, ELMO1, and ST7 were used.

Fig. 7.

FBXL11 gene is induced upon NF-κB activation. (A) Western analysis, showing that FBXL11 expression is induced in 293C6 cells on IL-1β treatment (Left) but is blocked in 293C6SR-κB cells, which express the dominant negative IκB superrepressor (Right). (B) Model. In addition to previously known regulatory pathways, NF-κB is regulated by the methylation and demethylation of p65, catalyzed by the NSD1-FBXL11 enzyme pair. FBXL11 gene is induced upon NF-κB activation, forming a negative regulatory loop.