Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB

Lü, Tao; Jackson, Mark W.; Singhi, Aatur D.; Kandel, Eugene; Yang, Maojing; Zhang, Yi; Gudkov, Andrei V.; Stark, George R.

doi:10.1073/pnas.0908560106

Cited by 74 publications

(106 citation statements)

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“…We tested whether NSD1, the known methylase for H3K36, also affects NF-κB activity, using human 293C6 and colon cancer HT29 cells, with either normal or greatly increased levels of FBXL11, and also Z3 cells, a 293C6-derived cell line with constitutive NF-κB activity (10), together with the SD1-11 mutant of Z3 cells in which FBXL11 is overexpressed (8). A κB-luciferase reporter was transfected transiently into the cells together with NSD1 and, 48 h later, luciferase assays were carried out, with β-galactosidase as an internal control.…”

Section: Fbxl11 and Nsd1 Have Opposite Effects On Nf-κb; Both Bind Tomentioning

confidence: 99%

“…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, previously shown to demethylate histone H3K36, contains an F-box, a JmjC domain, a CxxC zinc finger, a PHD domain, and three leucine-rich repeat elements (9).…”

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“…FBXL11, previously shown to demethylate histone H3K36, contains an F-box, a JmjC domain, a CxxC zinc finger, a PHD domain, and three leucine-rich repeat elements (9). The point mutation H212A, in the JmjC domain, abolishes both H3K36 demethylase activity (9) and the ability of FBXL11 to inhibit NF-κB function (8). Here, we show that FBXL11 and its partner H3K36 methylase NSD1 function as an enzyme pair to regulate NF-κB through reversible lysine methylation of K218 and K221 of the p65 subunit of NF-κB, with important physiological consequences.…”

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

Lü

Jackson²,

Wang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Fbxl11 and Nsd1 Have Opposite Effects On Nf-κb; Both Bind Tomentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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

Lü

Jackson²,

Wang

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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252

202

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“…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. 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.…”

Section: Lysine Methylation Of Nonhistone Proteinsmentioning

confidence: 99%

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

2010

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p53, NFκB, STAT3, and several other transcription factors are reversibly methylated on lysine residues by enzymes that also modify histones. The methylations of NFκB and STAT3 take place when they are bound to promoters, suggesting a more general model in which the binding of inducible transcription factors to DNA helps to recruit chromatin-modification machinery, which then may modify not only histones but also the bound transcription factors. Mutations of some histone-lysine methyltransferases and demethylases are linked to cancer, and these mutations may alter the methylation not only of histones but also of transcription factors, and thus may be tumorigenic through more than one mechanism. In recent years, much attention has been focused on posttranslational modification of chromatin because of its critical role in regulating gene expression [1]. The N-terminal tails of histones, as well as positions in the globular domains, carry methylations, acetylations, phosphorylations, ADP ribosylations, ubiquitinations, sumoylations, and other modifications [2]. Many different amino acid residues of each of the four core histones have been identified as modification sites [3] and some lysine side chains can be methylated or acetylated alternatively. These modifications provide entry sites for accessory proteins that help to determine higher order chromatin organization, leading to the activation or inactivation of specific genes. The lysine ε-amino groups can receive one, two, or three methyl groups and the extent of lysine methylation at a single lysine residue can be read differently by different effector proteins [4]. H3K4 methylation is associated with an early-elongating form of RNA polymerase II at actively transcribed genes, H3K9 and H3K27 methylation are linked to heterochromatin formation, while H3K36 methylation provides a stable molecular mechanism for establishing chromatin context throughout the genome by distinguishing potential regulatory regions from transcribed chromatin [5]. 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. Here, we provide a summary of recent work showing lysine methylation of transcription factors that have well-recognized roles in tumorigenesis and of the effects of mutations of lysine methyltransferases and demethylases in cancer, and we cite evidence that at least some modifications take place only on promoter-bound transcription factors. Lysine methylation of nonhistone proteinsReversible modification of histones by methylation and demethylation, which occur at both lysine and arginine residues, plays an important role in many biological processes, including transcri...

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“…The enzyme has been reported to be involved in the regulation of NF‐κB signalling17 and the control of stem‐cell differentiation and proliferation 18. Its overexpression in gastric and small‐cell lung cancer cells suggests that inhibiting KDM2A may represent a strategy for targeting certain cancers at the transcription level 19, 20.…”

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Discovery of a Highly Selective Cell‐Active Inhibitor of the Histone Lysine Demethylases KDM2/7

et al. 2017

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Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell‐permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure–activity relationships, leading to the development of a small molecule with around 75‐fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.

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Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB

Cited by 74 publications

References 24 publications

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

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

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

Discovery of a Highly Selective Cell‐Active Inhibitor of the Histone Lysine Demethylases KDM2/7

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