Primate TNF promoters reveal markers of phylogeny and evolution of innate immunity

Abstract

Background: Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry.

Methodology/principal findings: Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages.

Conclusions/significance: Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF.

Figure 1. The human TNF gene promoter.

The sequence of the human TNF promoter from −200 to +1 nt relative to the start site of transcription is shown, along with previously identified phylogenetic footprints and binding sites for the indicatedtranscription factors, including sites identified as binding sites for multiple factors (crosshatched) in quantitative DNase I footprinting assays, by EMSA analysis, and by chromatin immunoprecipitation assays –. The sequence of the upstream Sp1 site (upSp1) found in the indicated genera and species is shown, with fixed nucleotide differences from the human sequence indicated in red. The thymine at position −9, which is invariant in all non-human primates studied previously and in this study, is also shown in red.

Figure 2. Primate species examined in this study.

The common and taxonomic designation for each species or subspecies , , number of individuals from which DNA for sequence analysis was isolated, and the source of the sampled individuals are indicated. The region of the promoter corresponding to the upstream Sp1 site (Figure 1) was sequenced for Cercopithecus ascanius, Trachypithecus francoisi, Pygathrix nemaeus, and Lemur catta (Figure 7A). For all other species and subspecies, the complete proximal and distal TNF promoter regions (−1153 to +69 nt relative to the start site of transcription) were sequenced.

Figure 3. TNF promoter-based primate phylogenetic trees.

Extended majority rule consensus trees derived from 1000 replications by bootstrap analysis generated from a distance matrix between ∼1.2 kb of sequence for each taxon using the Kimura 2-parameter model (A) and maximum parsimony (B) is shown. Values at internodes indicate the number of times the group consisting of the species to the right of the internode occurred among the trees (out of 1000 trees).

Figure 4. Conservation of the proximal TNF promoter region in primates.

Multiple sequence alignment (MSA) of the TNF promoter (−1153 to +69 nt relative to the start site of transcription) for human and all other primate species examined in this study using eShadow . Nucleotide position relative to the TNF start site of transcription is on the x-axis. Percentage variation (inversely proportional to conservation) is on the y-axis. Peaks and valleys of the conservation plot correspond to regions of low and high variation, respectively, and 0% variation signifies 100% sequence identity in the MSA. Black shading indicates regions of highest sequence conservation.

Figure 5. Fixed differences in the TNF promoter distinguish primate species and subspecies.

Column headers indicate the position in the human TNF promoter relative to the start site of transcription. The corresponding nucleotide sequence, deletion, or insertion for each species is indicated for each row. Blue shading in a column indicates groups of primate subspecies within a species (or species within a genus) distinguished by a fixed difference.

Figure 6. Fixed differences in the TNF promoter mark primate ancestry.

Clade diagram illustrating the basic and approximate evolutionary relationships among the primate species in this study, with selected times of divergence (in millions of years) noted. Groups of fixed genetic differences (the total number in parenthesis) completely conserved within clades are marked at the appropriate branch points (see Table S3). Human TNF promoter SNPs which are ancestral in the primate lineage are listed. Ancestral sequences and transitions or transversions in the TNF promoter are noted at the appropriate branchpoints.

Figure 7. Functional analysis of fixed genetic differences in the upstream Sp1 site in primate TNF promoters.

A. Sequence of the upstream Sp1 site in the TNF promoter from humans and the indicated non-human primates, with the horse (Equus caballus) and pig (Sus scrofa) sequences for comparison. Nucleotide positions relative to the start site of TNF transcription are shown. B. EMSA of nuclear extracts from the indicated cell lines incubated with radiolabeled oligonucleotides corresponding to the indicated primate TNF upstream Sp1 site. Positions of the Sp1- and Sp3-DNA complexes, confirmed by antibody supershift assays (data not shown) are indicated. C. Transfections in the indicated cell lines with luciferase reporter genes fused to the proximal TNF promoter (−200 to +87 nt) of the indicated primate species. Fold induction of luciferase activity in response to treatment with ionomycin (I) in 68-41 T cells, treatment with lipopolysaccharide (LPS) in J774 monocytic cells, or infection with Mycobacterium tuberculosis (MTb) in J774 monocytic cells is shown.