Wade H. Powell

IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF TWO HIGHLY DIVERGENT ARYL HYDROCARBON RECEPTORS (AHR1 AND AHR2) IN THE TELEOST FUNDULUS HETEROCLITUS : EVIDENCE FOR A NOVEL SUBFAMILY OF LIGAND-BINDING BASIC HELIX LOOP HELIX- PER-ARNT-SIM (BHLH-PAS) FACTORS

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds cause altered gene expression and toxicity. The AHR belongs to an emerging multigene family of transcription factors possessing basic helix loop helix (bHLH) and Per-ARNT-Sim (PAS) domains. Most bHLH-PAS proteins occur as duplicates or “paralog groups” in mammals, but only a single mammalian AHR has been identified. Here we report the cDNA cloning of two distinct AHRs, designated FhAHR1 and FhAHR2, from a single vertebrate species, the teleost Fundulus heteroclitus (Atlantic killifish). Both Fundulus AHR proteins possess bHLH and PAS domains that are closely related to those of the mammalian AHR. FhAHR1 and FhAHR2 are highly divergent (40% overall amino acid identity; 61% identity in the N-terminal half), suggesting that they arose from a gene duplication predating the divergence of mammals and fish. Photoaffinity labeling with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin and velocity sedimentation analysis using 2,3,7,8-[1,6-3H]TCDD showed that both FhAHR1 and FhAHR2 exhibit specific, high-affinity binding of dioxins. Both AHRs also showed specific, TCDD- and ARNT-dependent interactions with a mammalian xenobiotic response element. The two Fundulus AHR genes displayed different tissue-specific patterns of expression; FhAHR1 transcripts were primarily expressed in brain, heart, ovary, and testis, while FhAHR2 transcripts were equally abundant in many tissues. Phylogenetic analysis demonstrated that Fundulus AHR1 is an ortholog of mammalian AHRs, while AHR2 forms in Fundulus and other fish are paralogous to Fundulus AHR1 and the mammalian AHRs and thus represent a novel vertebrate subfamily of ligand-binding AHRs.

An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and β-naphthoflavone binding

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.

PURIFICATION OF RNA POLYMERASE II GENERAL TRANSCRIPTION FACTORS FROM RAT LIVER

Publisher Summary Eukaryotic messenger RNA synthesis is a complex biochemical process requiring the concerted action of multiple general transcription factors (TFs) that control the activity of RNA polymerase II at both the initiation and elongation stages of transcription. Because the general transcription factors are present at low levels in mammalian cells, their purification is a formidable undertaking. This chapter explores the feasibility of using rat liver as a source for purification of the general factors. Rat liver has proven to be an ideal model system for biochemical studies of transcription initiation and elongation by RNA polymerase II. The chapter develops convenient and reproducible methods for preparation of rat liver extracts from as much as 1 kg of liver per day. Because it is both technically difficult and expensive to obtain such quantities of cultured cells on a daily basis, rat liver provides a significant logistic advantage for purification of the general transcription factors.

Responsiveness of a Xenopus laevis cell line to the aryl hydrocarbon receptor ligands 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Frogs are very insensitive to TCDD toxicity, and AHRs from Xenopus laevis (African clawed frog) bind TCDD with >20-fold lower affinity than mouse AHR(b-1). Frog AHRs may nonetheless be highly responsive to structurally distinct compounds, especially putative endogenous ligands. We sought to determine the responsiveness of an X. laevis cell line, XLK-WG, to the candidate endogenous AHR ligand 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct that exhibits high potency in mammalian systems. FICZ readily induced mRNAs for CYP1A6 and CYP1A7. Cells exposed to FICZ for 3h expressed up to 5-fold greater quantities of CYP1A6/7 mRNAs than those exposed for 24h, suggesting FICZ is metabolized following rapid enzyme induction. FICZ appeared more potent than TCDD. Following a 3-h exposure, the EC(50) for CYP1A6 mRNA induction by FICZ was approximately 6nM, while the TCDD response was greater than 174nM. These potencies were lower than those determined for mouse hepatoma cells (Hepa1c1c7; EC(50)= approximately 0.06nM each). The difference in ligand potency between cell lines was confirmed by induction of ethoxyresorufin-O-deethylase (EROD) activity. mRNA from XLK-WG cells treated with 100nM FICZ, 100nM TCDD, or vehicle was also analyzed on expression microarrays. FICZ altered the expression of 105 more transcripts than TCDD, and common targets were altered more dramatically by FICZ. Overall, these studies demonstrate that although FICZ is a less potent CYP1A inducer in frog cells than in mouse cells, the reduction is much less than for TCDD. Relative conservation of the FICZ response in a TCDD-insensitive species suggests its physiological importance as an AHR ligand.

The evolution of aryl hydrocarbon signaling proteins: diversity of ARNT isoforms among fish species.

The aryl hydrocarbon receptor nuclear translocator (ARNT) mediates aryl hydrocarbon signaling and toxicity by dimerizing with the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that binds specific DNA elements and alters transcription of target genes. Two genes encode different forms of ARNT in rodents: ARNT1, which is widely expressed, and ARNT2, which exhibits a very restricted expression pattern. In an effort to characterize aryl hydrocarbon signaling mechanisms in fishes, we previously isolated an ARNT cDNA from Fundulus heteroclitus and discovered that this species expresses ARNT2 ubiquitously. This situation differs not only from mammals, but also from rainbow trout, which expresses a divergent ARNT gene that we hypothesized was peculiar to salmonids (rtARNTa/b). In this communication, we examine the ARNT sequences of multiple fish species, including a newly isolated cDNA from scup (Stenotomus chrysops). Our phylogenetic analysis demonstrates that zebrafish ARNT, like the Fundulus protein, is an ARNT2. Contrary to expectations, the scup ARNT is closely related to the rainbow trout protein, demonstrating that the existence of this ARNT isoform predates the divergence of salmonids from the other teleosts. Thus, different species of fish express distinct and highly conserved isoforms of ARNT. The number, type, and expression pattern of ARNT proteins may contribute to interspecies differences in aryl hydrocarbon toxicity, possibly through distinct interactions with additional PAS-family proteins.

An aryl hydrocarbon receptor repressor from Xenopus laevis: function, expression, and role in dioxin responsiveness during frog development.

Xenopus laevis and other frogs are extremely insensitive to the toxicity of xenobiotic ligands of the aryl hydrocarbon receptor (AHR), including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Premetamorphic life stages are especially insensitive, and they are reported to be refractory to induction of Cytochrome P4501As, which are readily induced in older animals. The AHR repressor (AHRR) is a member of the AHR gene family. AHRR expression is induced by TCDD; it then represses AHR in an apparent negative feedback loop. In this study, we sought to test the hypothesis that constitutive AHRR expression underlies the lack of TCDD responsiveness in frog early life stages. We determined the sequence of an AHRR complimentary DNA encoding an 85.3-kDa protein sharing 52-55% identity with the bHLH/PAS domains of other AHRRs. In transient transfection assays, X. laevis AHRR inhibited TCDD-induced reporter gene expression mediated by either X. laevis AHR paralog, AHR1alpha or AHR1beta. AHRR messenger RNA was expressed at low levels in embryos (Nieuwkoop-Faber stage 33-38; approximately 52 h.p.f.) and was induced approximately twofold following TCDD exposure (42 ng/g wet weight). In contrast, AHRR exhibited higher constitutive expression and was induced more than threefold in tadpoles at stage 52-55 (prometamorphic; approximately 4 weeks postfertilization) and in isolated viscera of stage 62 tadpoles (in the metamorphic climax; approximately 7 weeks postfertilization). Although the magnitude of induction was smaller, the temporal pattern of AHRR expression and inducibility resembled that of CYP1A6. Thus, attenuated transcriptional activation of AHR target genes and low TCDD toxicity in X. laevis embryos cannot be explained by constitutive, high-level expression of AHRR.

Specific Ligand Binding Domain Residues Confer Low Dioxin Responsiveness to AHR1β of Xenopus laevis

The aryl hydrocarbon receptor (AHR) is a Per-ARNT-Sim (PAS) family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs from Xenopus laevis bind TCDD with low affinity. We sought to identify structural features of X. laevis AHR1β associated with low TCDD sensitivity. Substitution of the entire ligand binding domain (LBD) with the corresponding sequence from mouse AHR(b-1) dramatically increased TCDD responsiveness in transactivation assays. To identify the amino acid residues responsible, we constructed a comparative model of the AHR1β LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity with dimensions similar to those of the putative binding cavity of mouse AHR(b-1), suggesting the importance of side chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved β-strand, was changed to serine, the corresponding mouse residue, the EC50 for TCDD decreased more than 15-fold. When N325 was changed to serine, the EC50 decreased 3-fold. When the mutations were combined, the EC50 decreased from 18.6 to 0.8 nM, the value nearly matching the TCDD sensitivity of mouse AHR. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased levels of TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ). Mutations that increased sensitivity to TCDD also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists.

Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis.

Cytochrome P450 family 1 (CYP1) includes four subfamilies of enzymes: CYP1A, CYP1B, CYP1C, and CYP1D. In many vertebrates, CYP1A, 1B, and 1C expression is induced by agonists of the aryl hydrocarbon receptor, including toxic contaminants such as chlorinated dioxins, coplanar chlorinated biphenyls, and polynuclear aromatic hydrocarbons. Assessed at the level of mRNA, protein, or enzyme activity, CYP1s (especially CYP1As) represent potent and popular biomarkers of contaminant exposure in aquatic vertebrates. Alkylated resorufins are synthetic substrates used to detect, quantify, and describe catalytic activities of cytochrome P450s. The ability to oxidize specific resorufin-based substrates can distinguish the catalytic activities of individual CYP1s. Xenopus laevis, the African clawed frog, is the most widely employed amphibian model in aquatic toxicology, yet the number, inducibility, and activities of CYP1s have not been systematically characterized in this species. Here we report the cloning of cDNAs encoding two new CYP1 family members, X. laevis CYP1B and CYP1C, along with an integrated assessment of the induction of alkyloxyuresorufin-O-dealkylase (AROD) activities and mRNA expression of four known X. laevis CYP1s: CYP1A6, CYP1A7, CYP1B, and CYP1C. Using XLK-WG, an X. laevis kidney epithelial cell line, we determined that EROD (ethoxyresorufin substrate) and MROD (methoxyresorufin) were both induced 3000- to 5000-fold following 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) exposure up to 250 nM, while BROD (benzyloxyresorufin) and PROD (pentyloxyresorufin) activity was not detectable regardless of TCDD treatment. TCDD induced CYP1A6 and CYP1A7 mRNAs by 2-3 orders of magnitude, while CYP1B and CYP1C were unchanged. The more potent AHR agonist, FICZ (6-formylindolo[3,2-b]carbazole), induced CYP1B up to 10-fold at concentrations between 0.1 and 250 nM, while CYP1C induction was less than 3-fold. CYP1B mRNA showed the highest constitutive mRNA expression, 5- to 75-fold greater than the other CYP1 transcripts. Taken together, these results suggest that CYP1A6 and CYP1A7 perform the bulk of EROD and MROD activities we observed in these cells. The ability of each X. laevis CYP1 to catalyze oxidation of individual resorufin substrates remains to be determined. Correlating CYP1 mRNA and induced AROD activity is a significant step toward clarifying the biochemical meaning of these biomarkers and the roles of CYP1 enzymes in X. laevis. The cell culture approach represents an important complement to the long standing use of frog embryos and tadpoles in toxicological studies, providing a well suited model system for determining the molecular mechanisms underlying the regulation of these important biomarkers of contaminant exposure.

Subfunctionalization of Paralogous Aryl Hydrocarbon Receptors from the Frog Xenopus Laevis: Distinct Target Genes and Differential Responses to Specific Agonists in a Single Cell Type

Gene duplication confers genetic redundancy that can facilitate subfunctionalization, the partitioning of ancestral functions between paralogs. We capitalize on a recent genome duplication in Xenopus laevis (African clawed frog) to interrogate possible functional differentiation between alloalleles of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates toxicity of dioxin-like compounds and plays a role in the physiology and development of the cardiovascular, hepatic, and immune systems in vertebrates. X. laevis has 2 AHR genes, AHR1&agr; and AHR1&bgr;. To test the hypothesis that the encoded proteins exhibit different molecular functions, we used TALENs in XLK-WG cells, generating mutant lines lacking functional versions of each AHR and measuring the transcriptional responsiveness of several target genes to the toxic xenobiotic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the candidate endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ). Mutation of either AHR1&agr; or AHR1&bgr; reduced TCDD induction of the canonical AHR target, Cytochrome P4501A6, by 75%, despite the much lower abundance of AHR1&bgr; in wild-type cells. More modestly induced target genes, encoding aryl hydrocarbon receptor repressor (AHRR), spectrin repeat-containing nuclear envelope protein 1 (SYNE-1), and gap junction protein gamma 1 (GJC1), were regulated solely by AHR1&agr;. AHR1&bgr; was responsible for CYP1A6 induction by FICZ, while AHR1&agr; mediated FICZ induction of AHRR. We conclude that AHR1&agr; and AHR1&bgr; have distinct transcriptional functions in response to specific agonists, even within a single cell type. Functional analysis of frog AHR paralogs advances the understanding of AHR evolution and as well as the use of frog models of developmental toxicology such as FETAX.

An aryl hydrocarbon receptor from the salamander ambystoma mexicanum exhibits low sensitivity to 2,3,7,8-tetrachlorodibenzo -p -dioxin

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1β (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.