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Stimulation of the NF-kappaB pathway often causes p65-p50 and p50-p50 dimers to be simultaneously present in the cell nucleus. A natural polymorphism at nucleotide -863 in the human TNF promoter (encoding tumor necrosis factor [TNF]) region provides an opportunity to dissect the functional interaction of p65-p50 and p50-p50 at a single NF-kappaB binding site. We found that this site normally binds both p65-p50 and p50-p50, but a single base change specifically inhibits p50-p50 binding. Reporter gene analysis in COS-7 cells expressing both p65-p50 and p50-p50 shows that the ability to bind p50-p50 reduces the enhancer effect of this NF-kappaB site. Using an adenoviral reporter assay, we found that the variant which binds p50-p50 results in a reduction of lipopolysaccharide-inducible gene expression in primary human monocytes. This finding adds to a growing body of experimental evidence that p50-p50 can inhibit the transactivating effects of p65-p50 and illustrates the potential for genetic modulation of inflammatory gene regulation in humans by subtle nucleotide changes that alter the relative binding affinities of different forms of the NF-kappaB complex.

Original publication

DOI

10.1128/mcb.20.24.9113-9119.2000

Type

Journal article

Journal

Molecular and cellular biology

Publication Date

12/2000

Volume

20

Pages

9113 - 9119

Addresses

Molecular Infectious Disease Group, Institute of Molecular Medicine, Oxford University, Oxford, United Kingdom. iudalova@molbiol.ox.ac.uk

Keywords

Monocytes, Cells, Cultured, Cell Line, COS Cells, Macrophages, Animals, Humans, Adenoviridae, Lipopolysaccharides, Tumor Necrosis Factor-alpha, NF-kappa B, Cell Fractionation, Cloning, Molecular, Gene Expression Regulation, Binding Sites, Dimerization, Polymorphism, Genetic, Genes, Reporter, NF-kappa B p50 Subunit, Promoter Regions, Genetic, Transcriptional Activation