Search

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The recently identified AHR repressor (AHRR) forms a negative feedback loop with the AHR. We investigated AHRR structure, function, evolution, and regulation in zebrafish, a powerful model in developmental biology and toxicology. We identified and cloned two distinct AHRR cDNAs that encode predicted proteins of 550 (AHRR1) and 573 (AHRR2) amino acids. The ahrr1 and ahrr2 genes map to zebrafish chromosomes 24 and 2, respectively, both of which share conserved synteny with human chromosome 5, the location of human AHRR. Mapping and phylogenetic analysis show that AHRR1 and AHRR2 are co-orthologs of the mammalian AHRR. In transient transfection assays, AHRR1 and AHRR2 repressed constitutive and TCDD-inducible transactivation by AHR2. Expression of both AHRR mRNAs was induced in ZF-L cells by AHR agonists but not by non-agonists. TCDD induced AHRR1 and AHRR2 expression in a dose-dependent manner in ZF-L cells, with EC50 values similar to those for induction of CYP1A. Both AHRRs were expressed and induced by TCDD in zebrafish embryos. Thus, zebrafish possess duplicate AHR-regulated AHRR paralogs that act in a negative feedback loop to repress the AHR signaling pathway.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the bHLH/PAS protein superfamily. ARNT dimerizes with several PAS superfamily members, including the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that alters transcription by binding specific elements within the promoters of target genes. Two genes encode different forms of the protein in rodents: ARNT1, which is widely expressed, and ARNT2, which is limited to the brain and kidneys of adults and specific neural and branchial tissues of embryos. In an effort to characterize aryl hydrocarbon signaling mechanisms in Fundulus heteroclitus, a marine teleost that can develop heritable xenobiotic resistance, we have isolated a liver cDNA encoding an ARNT homolog. The protein exhibits AHR-dependent DNA binding capability typical of other vertebrate ARNTs. Unexpectedly, phylogenetic analysis reveals that the cDNA encodes an ARNT2. This is the only detectable ARNT sequence in Fundulus liver, gill, ovary, and brain, suggesting that ARNT2 is the predominant form of ARNT in this species. Also surprising is the relative lack of sequence identity with another fish ARNT protein, rainbow trout ARNTb, which we show forms a distinct branch outside the ARNT1 and ARNT2 clades in phylogenetic analyses. Functional diversity of ARNT proteins in fish may have important implications for the assessment of aryl hydrocarbon effects on natural populations. The increasing use of fish models in developmental and toxicological studies underscores the importance of identifying taxon-specific roles of ARNT proteins and their potential dimeric partners in the PAS superfamily., (Copyright 1999 Academic Press.)