Complex NF-κB Interactions at the Distal Tumor Necrosis Factor Promoter Region in Human Monocytes

Abstract

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 B (NF-B)-like motifs within the human TNF promoter region. The area contains two NF-B 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-B as well as an unknown protein(s) of approximately 40 kDa. We show that these three adjacent B-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-B heterodimers simultaneously. Reporter gene studies indicate that each element of the cluster contributes to lipopolysaccharideinduced transcriptional activation in Mono Mac 6 cells. These findings suggest that NF-B acts in a complex manner to activate TNF transcription in human monocytes.A fundamental problem in infectious disease is to understand the mechanisms by which microbial stimuli such as bacterial lipopolysaccharide (LPS) 1 regulate the expression of human inflammatory mediators such as tumor necrosis factor-␣ (TNF). Production of TNF by macrophages and monocytes occurs early in infection and orchestrates much of the subsequent inflammatory response (1). Because excessive production of TNF is responsible for many of the pathological consequences of severe infection, its regulation is critical. This occurs at both the transcriptional and post-transcriptional levels (2), with transcriptional regulation showing specificity for both stimulus and cell type (3). Reporter gene studies of the human TNF promoter using a range of stimuli and cell types have identified a number of regulatory elements upstream of the transcriptional start site, mostly within the proximal 200 base pairs (4 -13). However, we still have a poor understanding of the precise regulatory events that are responsible for the rapid induction of high level TNF expression in human monocytes and macrophages during acute bacterial infection.Much debate has centered on the role of nuclear factor-B (NF-B) in transcriptional activation of the human TNF gene in monocytes. Although there is strong evidence that murine TNF expression is influenced by a number of NF-B binding sites (14 -16), the situation in humans is less clear. Several lines of circumstantial evidence suggest that NF-B could play an important role. A number of NF-B-like binding motifs are located between the start of transcription of the TNF gene and the 3Ј terminus of the lymphotoxin-␣ gene: they are here denoted B1 (Ϫ873 to Ϫ864 nt), B2 (Ϫ627 to Ϫ618 nt), B2a (Ϫ598 to Ϫ589 nt), CK-1 (Ϫ213 to Ϫ204 nt (17)), and B3 (Ϫ98 to Ϫ89 nt). It is worth noting that site B2...

TNF-α Gene Expression in Macrophages: Regulation by NF-κB Is Independent of c-Jun or C/EBPβ

“…As early as 20 min following stimulation with LPS, NF-B p65/p50 heterodimers bound to the B1 and B3 binding sites of the proximal TNF-␣ promoter, consistent with earlier studies (7,8). The current study extends these observations by directly documenting the functional relevance of these heterodimers, because expression of NF-B p65 and p50 resulted in greater than additive, or synergistic, activation of the TNF-␣ promoter.…”

“…We previously demonstrated that the Ϫ100/Ϫ74 oligonucleotide binds ectopically expressed C/EBP␤ (5,22) and it possesses the B3 binding site (8). Constitutively, there was minimal binding of NF-B to this oligonucleotide (Fig.…”

“…Oligonucleotide probes spanning the following regions of the TNF-␣ promoter were employed: Ϫ100 to Ϫ74 bp (Ϫ100/Ϫ74), possessing the B3 (Ϫ98 to Ϫ89) and the C/EBP␤ binding sites; Ϫ115 to Ϫ98 bp (Ϫ115/Ϫ98), possessing the AP-1 binding site; Ϫ598 to Ϫ589 bp (Ϫ598/Ϫ589) representing the B1 binding site; and Ϫ217 to Ϫ200 bp (Ϫ217/Ϫ200) representing the B2 binding site (7,8,26). In addition, oligonucleotides representing the C/EBP␤ binding site of the IL-6 promoter and an AP-1 binding site from the collagenase promoter have been previously described (22).…”

Is there a future for TNF promoter polymorphisms?

“…Most of the factors that have been convincingly described as being involved in TNF transcription, including CREB, 157 C/EBPa þ b, [158][159][160] NFATp, [161][162][163] SP-1, Egr-1, 164 ATF2/JUN, 162,163,165,166 Nrf-1, 167,168 and Ets, 169 bind within the 200 bp proximal promoter. Although functional binding sites in the À600 region have been reported for NF-kB 170,171 and Litaf, 172,173 these regions harbour no known SNP. These considerations place a heavy burden of proof on data claiming to show functional activity outside of the 'core' promoter, or other regions for which there is independent evidence of protein binding.…”

Increased in vivo transcription of an IL-8 haplotype associated with respiratory syncytial virus disease-susceptibility

“…35 The binding reaction and supershift experiments using antibodies raised against C/EBP b, Oct-1 and p65 NFkB (Santa Cruz Biotechnology, Santa Cruz, USA) were performed as described. 36 Statistics Data are summarized means with standard errors. The significance of differences in mean values, where the data had a normal distribution, was determined using a paired t-test.…”