Abstract
The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds occur via the aryl hydrocarbon receptor (AHR), a member of the basic helix-loop-helix-Per-ARNT-Sim homology (bHLH-PAS) protein superfamily. A single AHR gene has been identified in mammals, whereas many fish species, including the Atlantic killifish (Fundulus heteroclitus) possess two distinct AHR genes (AHR1 and a novel form, AHR2). A mouse bHLH-PAS protein closely related to AHR and designated AHR repressor (AHRR) is induced by 3-methylcholanthrene and represses the transcriptional activity of the AHR. To determine whether AHRR is the mammalian ortholog of fish AHR2 and to investigate the mechanisms by which AHRR regulates AHR function, we cloned an AHRR ortholog in F. heteroclitus with high sequence identity to the mouse and human AHRRs. Killifish AHRR encodes a 680-residue protein with a predicted molecular mass of 75.2 kDa. We show that in vitro expressed AHRR proteins from human, mouse, and killifish all fail to bind [(3)H]TCDD or [(3)H]beta-naphthoflavone. In transient transfection experiments using a luciferase reporter gene under control of AHR response elements, killifish AHRR inhibited the TCDD-dependent transactivation function of both AHR1 and AHR2. AHRR mRNA is widely expressed in killifish tissues and is inducible by TCDD or polychlorinated biphenyls, but its expression is not altered in a population of fish exhibiting genetic resistance to these compounds. The F. heteroclitus AHRR promoter contains three putative AHR response elements. Both AHR1 and AHR2 activated transcription of luciferase driven by the AHRR promoter, and AHRR could repress its own promoter. Thus, AHRR is an evolutionarily conserved, TCDD-inducible repressor of AHR1 and AHR2 function. Phylogenetic analysis shows that AHRR, AHR1, and AHR2 are distinct genes, members of an AHR gene family; these three vertebrate AHR-like genes descended from a single invertebrate AHR.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF443441 and AF443442
Are fish AHR2 and mammalian AHR repressor (AHRR) orthologous genes or do fish possess an AHRR gene? Is AHRR capable of repressing the function of both AHR1 and AHR2? Are both AHR1 and AHR2 involved in the regulation of AHRR expression? Is AHRR capable of high affinity binding of TCDD and other aromatic compounds that are known ligands for aryl hydrocarbon receptor (AHR)/AHR1 and AHR2 [1, 17]? Does enhanced expression of AHRR occur in fish selected for genetic resistance to TCDD?
We show (i) that killifish AHRR expression is inducible by TCDD and by PCBs in a variety of tissues in vivo, (ii) that this occurs through AHR response elements (AHREs) sequences in its promoter and can be mediated by either AHR1 or AHR2, and (iii) that the killifish AHRR is able to repress the function of its own promoter
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF443441 and AF443442. We show (i) that killifish AHRR expression is inducible by TCDD and by PCBs in a variety of tissues in vivo, (ii) that this occurs through AHRE sequences in its promoter and can be mediated by either AHR1 or AHR2, and (iii) that the killifish AHRR is able to repress the function of its own promoter. AHRR expression, like that of other AHR-regulated genes such as CYP1A1, is not induced in fish selected for genetic resistance to TCDD. We demonstrate that neither killifish AHRR, mouse AHRR, nor human AHRR is able to support high affinity binding of the AHR ligands [3H]TCDD or [3H]BNF, suggesting that the repressive function of this protein is ligandindependent. Our findings reveal that AHRR is one of three members of the vertebrate AHR gene family, which arose by duplication and divergence of a single ancestral AHR gene
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