Complex NF-kappaB interactions at the distal tumor necrosis factor promoter region in human monocytes.
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.
Oxford University Department of Paediatrics, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. iudalova@worf.molbiol.ox.ac.uk
I A
IA
Udalova
J C
JC
Knight
V
V
Vidal
S A
SA
Nedospasov
D
D
Kwiatkowski
D001483:Base Sequence; D002460:Cell Line; D018983:DNA Footprinting; D017931:DNA Primers; D019281:Dimerization; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D017931:DNA Primers; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha