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We describe a dense cluster of DNA-protein interactions located 600 nucleotides upstream of the transcriptional start site of the human tumor necrosis factor (TNF) gene. This area was identified as being of potential importance for lipopolysaccharide-inducible TNF expression in the human monocyte cell line Mono Mac 6, based on reporter gene analysis of point mutations at a number of nuclear factor kappaB (NF-kappaB)-like motifs within the human TNF promoter region. The area contains two NF-kappaB sites, which are here shown by DNase I and methylation interference footprinting to flank a novel binding site. UV cross-linking studies reveal that the novel site can also bind NF-kappaB as well as an unknown protein(s) of approximately 40 kDa. We show that these three adjacent kappaB-binding sites differ markedly in their relative affinities for p50/p50, p65/p65, and p65/p50, yet this 39-nucleotide segment of DNA appears capable of binding up to three NF-kappaB heterodimers simultaneously. Reporter gene studies indicate that each element of the cluster contributes to lipopolysaccharide-induced transcriptional activation in Mono Mac 6 cells. These findings suggest that NF-kappaB acts in a complex manner to activate TNF transcription in human monocytes.

Original publication

DOI

10.1074/jbc.273.33.21178

Type

Journal article

Journal

J Biol Chem

Publication Date

14/08/1998

Volume

273

Pages

21178 - 21186

Keywords

Base Sequence, Cell Line, DNA Footprinting, DNA Primers, Dimerization, Humans, Monocytes, NF-kappa B, Point Mutation, Protein Binding, Transcriptional Activation, Tumor Necrosis Factor-alpha