Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation

Abstract

Tumor necrosis factor alpha (TNF alpha) is a potent immunomodulator and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNF alpha are positively correlated with severity and mortality in malaria and leishmaniasis. We have previously described a polymorphism at -308 in the TNF alpha promoter and shown that the rare allele, TNF2, lies on the extended haplotype HLA-A1-B8-DR3-DQ2, which is associated with autoimmunity and high TNF alpha production. Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. Here we demonstrate, with reporter genes under the control of the two allelic TNF promoters, that TNF2 is a much stronger transcriptional activator than the common allele (TNF1) in a human B cell line. Footprint analysis using DNase I and B cell nuclear extract showed the generation of a hypersensitive site at -308 and an adjacent area of protection. There was no difference in affinity of the DNA-binding protein(s) between the two alleles. These results show that this polymorphism has direct effects on TNF alpha gene regulation and may be responsible for the association of TNF2 with high TNF alpha phenotype and more severe disease in infections such as malaria and leishmaniasis.

Figures

Figure 1

Induction of CAT protein from reporter gene constructs transfected into Raji cells. Raji cells (0.8 × 107) were transfected with the pBLCAT3 vector containing either no insert, which served as a negative control, or 691 bp of each allelic promoter. After 24 hr the cells were split in two, and one flask of each duplicate was stimulated with PMA (50 ng/ml). After a further 48 hr incubation the cells were harvested. Results have been corrected for transfection efficiency by Southern blot analysis of CAT DNA and also for total cell numbers by measuring total protein. The experiments were performed four times and means and standard errors are shown.

Figure 2

Specific binding of the TNF promoter fragment by a nuclear protein. Nuclear extract (10 μg) from Raji cells was incubated with each of the two TNF promoter fragments with (lanes 3–5 and 8–10) or without (lanes 1, 2, 6, 7) 100-fold molar excess of unlabeled probe. The gel retardation complex is indicated by “Protein/DNA complex”. Lanes 4 and 9, competition with unlabeled IL-1 DNA fragment did not disrupt the complex.

Figure 3

Detection of a hypersensitive site at −308. In vitro DNase I footprint analysis of TNF1 allele (coding strand) is shown. The unfilled arrowhead indicates an area of protection, and the filled arrowhead a DNase I hypersensitive site. Lanes: 1, Maxam–Gilbert guanidine ladder; 2, naked DNA control; 3, plus Raji cell nuclear extract.

Figure 4

Competitive EMSA using allelic TNF promoter fragments and Raji nuclear extracts. Labeled TNF2 was used. Lanes: 1 and 7, no competitor; lanes 2–6 and 7–12, increasing excess of unlabeled TNF2 and TNF1, respectively, as competitor. No difference in affinity for nuclear proteins between TNF alleles was apparent.