Complex NF-kappaB interactions at the distal tumor necrosis factor promoter region in human monocytes

J Biol Chem. 1998 Aug 14;273(33):21178-86. doi: 10.1074/jbc.273.33.21178.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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


  • DNA Primers
  • NF-kappa B
  • Tumor Necrosis Factor-alpha