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

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

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10-6 or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFκB. High levels of FBXL11 block the ability of NFκB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFκB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFκB-dependent signaling.

Original languageEnglish (US)
Pages (from-to)16339-16344
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume106
Issue number38
DOIs
StatePublished - Sep 22 2009
Externally publishedYes

Fingerprint

Insertional Mutagenesis
Leucine
Lysine
Histone Demethylases
Proteins
DNA
GTP-Binding Proteins
Mutagenesis
Virion
Small Interfering RNA
Organism Cloning
Clone Cells
Viruses
Gene Expression
Mutation
Infection
Population

Keywords

  • Cre recombinase
  • Lentivirus
  • Promoter insertion

ASJC Scopus subject areas

  • General

Cite this

Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB. / Lu, Tao; Jackson, Mark W.; Singhi, Aatur D.; Kandel, Eugene S.; Yang, Maojing; Zhang, Yi; Gudkov, Andrei V.; Stark, George R.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 106, No. 38, 22.09.2009, p. 16339-16344.

Research output: Contribution to journalArticle

Lu, Tao ; Jackson, Mark W. ; Singhi, Aatur D. ; Kandel, Eugene S. ; Yang, Maojing ; Zhang, Yi ; Gudkov, Andrei V. ; Stark, George R. / Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFκB. In: Proceedings of the National Academy of Sciences of the United States of America. 2009 ; Vol. 106, No. 38. pp. 16339-16344.
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