COMPLEX NF-KAPPA-B INTERACTIONS AT THE DISTAL TUMOR-NECROSIS-FACTOR PROMOTER REGION IN HUMAN MONOCYTES

We describe a dense cluster of DNA-protein interactions located 600 nu cleotides upstream of the transcriptional start site of the human tumo r necrosis factor (TNF) gene. This area was identified as being of pot ential importance for lipopolysaccharide-inducible TNF expression in t he human monocyte cell line Mono Mac 6, based on reporter gene analysi s of point mutations at a number of nuclear factor kappa B (NF-kappa B )-like motifs within the human TNF promoter region. The area contains two NF-kappa B sites, which are here shown by DNase I and methylation interference footprinting to flank a novel binding site. UV cross-link ing studies reveal that the novel site can also bind NF-kappa B as wel l as an unknown protein(s) of approximately 40 kDa. We show that these three adjacent kappa B-binding sites differ markedly in their relativ e affinities for p50/p50, p65/p65, and p65/p50, yet this 39-nucleotide segment of DNA appears capable of binding up to three NF-kappa B hete rodimers simultaneously. Reporter gene studies indicate that each elem ent of the cluster contributes to lipopolysaccharide-induced transcrip tional activation in Mono Mac 6 cells. These findings suggest that NF- kappa B acts in a complex manner to activate TNF transcription in huma n monocytes.