Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Figure 1

Alignment of PAS domain amino acid sequences of vertebrate AHRs and a possible invertebrate AHR homolog. Deduced amino acid sequences in the PAS domains of vertebrate AHRs and C. elegans C41G7.5 were aligned by using clustalw(1.6). GenBank accession numbers are listed in Table 2. Amino acids that are identical in five or more of the sequences are boxed and shaded. Similar amino acids are in boldface type. The PAS “A” and “B” imperfect repeats (as defined originally in refs. and 18) are underlined. The consensus sequence (≥50%) is shown below the aligned sequences.

Figure 2

Phylogenetic analysis of vertebrate AHR proteins. Gene trees were inferred from the amino acid alignment in Fig. 1 by using distance (NJ) (A and C) or maximum parsimony methods (B and D). The trees in A and B are unrooted, whereas in C and D the C. elegans C41G7.5 sequence was used as an outgroup (see Fig. 3 and text for explanation). (A and C) Distance (NJ) trees. Positions with gaps were excluded and corrections were made for multiple substitutions. Numbers in boldface type next to branch points are bootstrap values based on 1,000 samplings. The distance between sequences is the sum of the horizontal distances separating them. (B and D) Maximum parsimony trees. Exhaustive searches were performed by using paup 3.1.1 (33). The tree shown in B is the strict consensus of the three shortest trees (177 steps), based on 61 informative characters. The tree shown in D is the strict consensus of the six shortest trees (194–196 steps), based on 72 informative characters. In D, the shortest tree that could be constructed with the AHR2s (fish only) and all other AHRs (mammals and fish) as two monophyletic groups was 202 steps. In B and D, the length of the branches does not correspond to distance between the sequences.

Figure 3

Phylogenetic analysis of PAS family proteins. (A) Distance (NJ) tree. The tree was inferred from an alignment of PAS domains of all PAS proteins; the alignment is available upon request to M.E.H. or can be viewed at http://www.whoi.edu/biology/hahnm.html. GenBank accession numbers are listed in Table 2. Positions with gaps were excluded and no corrections were made for multiple substitutions. The bacterial kinA sequence was treated as the outgroup. Numbers in boldface type next to branch points are bootstrap values based on 1,000 samplings (values <50% are not shown). (B) Maximum parsimony tree. An heuristic search was performed by using paup 3.1.1 (33). The tree shown is the 50% majority rule tree, based on 163 informative characters. Numbers in boldface type next to branch points are bootstrap values from 100 samplings. Where multiple names are shown for synonymous proteins, the first name is that of the sequence used in the alignment.