Enhancers and transcription factors controlling the inducibility of the tumor necrosis factor-alpha promoter in primary macrophages.
In macrophages, the TNF-alpha promoter is specifically induced by bacterial endotoxin, and provides a good model for gene regulation during bacterial infections. We have analyzed the protein-binding characteristics and enhancer activity of four kappa B-like enhancers and of a MHC class II-like Y box found in the mouse TNF-alpha promoter. In addition to members of the NF-kappa B/rel transcription factor family, at least two of the kappa B sites also bound a nuclear protein identified as NF-GMa, a factor that binds to promoter sequences from many cytokines. When inserted upstream of an enhancer-less promoter, two of the kappa B sites were active as LPS-inducible enhancers in primary macrophages, whereas the other two were not. Mutations in nucleotides known to contact nuclear factors severely reduced affinity of the kappa B sites for NF-kappa B. Introduction of the same mutations into a construct containing 1059 bp of the TNF-alpha promoter coupled to a CAT reporter gene resulted in a stepwise reduction in inducibility by LPS; mutation of all four sites (11 bp of 1059) reduced inducibility by 90%, providing compelling evidence for the role of transcription factors belonging to the NF-kappa B/rel family in the activation of the TNF-alpha promoter. The TNF-alpha Y box bound an abundant nuclear factor, but had no detectable activity in our assays, either as an enhancer or as a mutation-sensitive controlling element.
Drouet
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Ludwig Institute for Cancer Research, Epalinges, Switzerland.
Shakhov
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Jongeneel
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D000818:Animals; D001483:Base Sequence; D004742:Enhancer Elements, Genetic; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D016179:Granulocyte Colony-Stimulating Factor