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

Abstract

NF-κB, a central coordinator of immune and inflammatory responses, must be tightly regulated. We describe a NF-κB regulatory pathway that is driven by reversible lysine methylation of the p65 subunit, carried out by a lysine methylase, the nuclear receptorbinding SET domain-containing protein 1 (NSD1), and a lysine demethylase, F-box and leucine-rich repeat protein 11 (FBXL11). Overexpression of FBXL11 inhibits NF-κB activity, and a high level of NSD1 activates NF-κB and reverses the inhibitory effect of FBXL11, whereas reduced expression of NSD1 decreases NF-κB activation. The targets are K218 and K221 of p65, which are methylated in cells with activated NF-κB. 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-κB, revealing a negative regulatory feedback loop. We conclude that reversible lysine methylation of NF-κB is an important element in the complex regulation of this key transcription factor. [RelA (p65), RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52)], the p65/p50 heterodimer functions most often in the "classical" signaling pathway (1). Constitutive NF-κB activation is frequently associated with a number of pathological conditions, including inflammation and cancer, and is well known to be involved in tumor angiogenesis and invasiveness (2). Loss of the normal regulation of NF-κB is a major contributor to the deregulated growth, resistance to apoptosis, and propensity to metastasize observed in many different cancers (3). Our data, showing NF-κB activation in many cancer-derived cell lines (4, 5), further support the strong correlation between constitutive NF-κB activation and cancer. Thus, understanding the complexity of NF-κB-dependent signaling is an important aspect of dealing with these diseases.NF-κB has a variety of functions and is regulated by hundreds of different stimuli in different cell types (6). Specialized control of NF-κB activity is critical for achieving its normal transient activation in response to stress. Regardless of the stimulus, all pathways leading to NF-κB activation depend on inducing posttranslational modifications of the IκB kinase, IκB proteins, or NF-κB subunits. These regulatory modifications, including phosphorylation, ubiquitination, acetylation, sumolation, and nitrosylation, vary depending on the specific stimulus and context (7).We have developed a lentiviral validation-based insertional (VBIM) method to create mutant cell lines in which the overexpression of specific cellular proteins, driven by inserted CMV promoters, is responsible for altered phenotypes. We used this method to identify the F-box and leucine-rich repeat protein 11 (FBXL11) as a potent negative regulator of NF-κB and to show that its demethylase activity was required for this function (8). FBXL11, previous...

A demethylation deficient isoform of the lysine demethylase KDM2A interacts with pericentromeric heterochromatin in an HP1a-dependent manner

“…10,14,[16][17][18][19] KDM2A also demethylates lysine residues of non-histone proteins such as the NF-kB p65 subunit or b-catenin. 20,21 KDM2A has been found to be misregulated in various cancers and its loss-of-function mouse mutants are embryonically lethal. 16,17,22 As opposed to the full length "long form" KDM2A protein (KDM2A-LF), which contains all the functional domains, the "short form" KDM2A protein (KDM2A-SF), lacks the N-terminal JmjC demethylation domain and therefore it is unable to function as a demethylase.…”

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

“…Three studies of NFκB and one of STAT3 showed that methylations of these transcription factors take place on promoters in the context of chromatin. 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.…”

“…Our laboratory has found that, in response to an activating signal such as treatment with IL-1, the p65 subunit of NFκB is inducibly methylated and demethylated on two specific lysine residues by chromatin-remodeling enzymes in ways that profoundly affect its function [7]. The activating monomethylation of K218 and dimethylation of K221 are both catalyzed by an H3K36 methylase, nuclear receptor-binding SET domain-containing protein 1 (NSD1); and these methyl groups are removed by an H3K36 demethylase, F-box leucine repeat rich protein 11 (FBXL11), leading to inactivation of NFκB.…”

“…The endogenous levels of some methylases and demethylases are also subject to change, for example, the gene encoding the demethylase FBXL11 is activated by NFκB [7] and the genes encoding the demethylases JMJD1A and JMJD2B are induced by HIF-1α in response to hypoxia [41]. The latter example and the work of Li et al [42] exemplify studies in which the levels of histone methylases and demethylases are changed, with biological consequences that have been interpreted in terms of altered histone modification.…”

“…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.…”