Identification of phylogenetic footprints in primate tumor necrosis factor-alpha promoters

The human tumor necrosis factor-alpha (TNF-alpha) gene encodes a pleiotropi c cytokine that plays a critical role in basic immunologic processes. To in vestigate the TNF-alpha regulatory region in the primate lineage, we isolat ed TNF-alpha promoters from representative great apes, old World monkeys, a nd New World monkeys. We demonstrate that there is a nonuniform distributio n of fixed human differences in the TNF-alpha promoter. We define a "fixed human difference" as a site that is not polymorphic in humans, but which di ffers in at least one of the seven primate sequences examined. Furthermore, we identify two human TNF-alpha promoter single nucleotide polymorphisms t hat are putative ancestral polymorphisms, because each of the human polymor phic nucleotides was found at the identical site in at least one of the oth er primate sequences. Strikingly, the largest conserved region among the pr imate species, a 69-nt "phylogenetic footprint." corresponds to a region of the human TNF-alpha promoter that forms the transcriptionally active nucle oprotein-DNA complex, essential for gene regulation. By contrast, other reg ions of the TNF-alpha promoter, which exhibit a high density of variable si tes, are nonessential for gene expression, indicating that distinct TNF-alp ha promoter regions have been subjected to different evolutionary constrain ts depending on their function. TNF-alpha is the first case in which a prom oter region dissected by functional analyses can be correlated with nucleot ide polymorphism and variability in primate lineages. The results suggest t hat patterns of polymorphism and divergence are likely to be useful in iden tifying candidate regions important for gene regulation in other immune-res ponse genes.