The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates gene expression following activation by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) or a variety of other synthetic and natural compounds. Previous studies have identified two AHR genes, AHR1 and AHR2, in zebrafish (Danio rerio), a widely used model species for studying vertebrate development and an emerging model in developmental toxicology. Zebrafish AHR2 binds TCDD with high affinity, is transcriptionally active and has a major role in mediating the developmental toxicity of TCDD. Zebrafish AHR1 lacks the ability to bind TCDD and activate transcription, and has no known function. In the present study, we report a new zebrafish AHR, designated AHR1B, which shares 34% amino acid sequence identity with AHR1 (AHR1A). The ahr1b gene resides on chromosome 22, adjacent to ahr2, whereas the ahr1a gene is located on chromosome 16. AHR1B is expressed in embryos as early as 24 hours post-fertilization and increases through the next 2 days, but expression is not inducible by TCDD. In contrast with the previously identified AHR1A, in vitro-expressed AHR1B protein exhibits specific, high-affinity binding of [³H]TCDD. Furthermore, AHR1B is able to activate the transcription of a reporter gene under the control of AHR response elements with an efficacy comparable with that of AHR2, but with a higher EC₅₀. We speculate that AHR1B may have a physiological role, such as in embryonic development, whereas AHR2 mediates the response to xenobiotics.

Background: The non-migratory killifish Fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the Atlantic coast of North America. Several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls (PCBs). Previous studies suggest that the mechanism of resistance to these and other “dioxin-like compounds” (DLCs) may involve reduced signaling through the aryl hydrocarbon receptor (AHR) pathway. Here we investigated gene diversity and evidence for positive selection at three AHR-related loci (AHR1, AHR2, AHRR) in F. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern US, including extremely polluted and reference estuaries, with a focus on New Bedford Harbor (MA, USA), a PCB Superfund site, and nearby reference sites. Results: We identified 98 single nucleotide polymorphisms within three AHR-related loci among all populations, including synonymous and nonsynonymous substitutions. Haplotype distributions were spatially segregated and F-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other F. heteroclitus loci. Genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. However, for AHR2 the New Bedford Harbor population had significant F_ST values in comparison to the nearest reference populations. Tests for positive selection revealed ten nonsynonymous polymorphisms in AHR1 and four in AHR2. Four nonsynonymous SNPs in AHR1 and three in AHR2 showed large differences in base frequency between New Bedford Harbor and its reference site. Tests for isolation-by-distance revealed evidence for non-neutral change at the AHR2 locus. Conclusion: Together, these data suggest that F. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. However, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in AHR1 and AHR2, and specific AHR2 SNPs and haplotypes that are associated with the PCB-resistant phenotype in the New Bedford Harbor population. The results suggest that AHRs, and especially AHR2, may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants.

Background: Populations of Atlantic killifish (Fundulus heteroclitus) have evolved resistance to the embryotoxic effects of polychlorinated biphenyls (PCBs) and other halogenated and nonhalogenated aromatic hydrocarbons that act through an aryl hydrocarbon receptor (AHR)-dependent signaling pathway. The resistance is accompanied by reduced sensitivity to induction of cytochrome P450 1A (CYP1A), a widely used biomarker of aromatic hydrocarbon exposure and effect, but whether the reduced sensitivity is specific to CYP1A or reflects a genome-wide reduction in responsiveness to all AHR-mediated changes in gene expression is unknown. We compared gene expression profiles and the response to 3,3',4,4',5-pentachlorobiphenyl (PCB-126) exposure in embryos (5 and 10 dpf) and larvae (15 dpf) fromF. heteroclituspopulations inhabiting the New Bedford Harbor, Massachusetts (NBH) Superfund site (PCB-resistant) and a reference site, Scorton Creek, Massachusetts (SC; PCB-sensitive).Results: Analysis using a 7,000-gene cDNA array revealed striking differences in responsiveness to PCB-126 between the populations; the differences occur at all three stages examined. There was a sizeable set of PCB-responsive genes in the sensitive SC population, a much smaller set of PCB-responsive genes in NBH fish, and few similarities in PCB-responsive genes between the two populations. Most of the array results were confirmed, and additional PCB-regulated genes identified, by RNA-Seq (deep pyrosequencing).Conclusions: The results suggest that NBH fish possess a gene regulatory defect that is not specific to one target gene such as CYP1A but rather lies in a regulatory pathway that controls the transcriptional response of multiple genes to PCB exposure. The results are consistent with genome-wide disruption of AHR-dependent signaling in NBH fish.

To test the relative induction of CYP1A1 and CYP1A2 in guinea pig adrenal and liver, the effects of 3,3′-methylcholanthrene (3MC), an archetypal inducer of CYP1A, were examined. RT-PCR analysis was performed using oligonucleotide primers corresponding to the 5′ and 3′ ends of the coding regions. PCR products of appropriate size (∼1.5kb) and sequence were obtained for CYP1A1 and CYP1A2 from both tissues; however, there were significant differences in the regulation of CYP1A expression in the liver vs adrenal. CYP1A1 mRNA was detectable in liver and adrenal of control guinea pigs and was induced in both tissues by 3MC, although levels were lower in the adrenal than in the liver. CYP1A2 mRNA was also present in both tissues of controls, but responded differently to 3MC in each. Strikingly, CYP1A2 mRNA decreased in adrenal, but increased in liver, following 3MC treatment. To begin to define mechanisms responsible for this differential regulation we compared levels of expression of AHR and ARNT, two transcription factors involved in the induction of CYP1A by aromatic hydrocarbons such as 3MC. RT-PCR analysis was performed using degenerate oligonucleotide primers designed against AHR and ARNT sequences from highly conserved regions of the 5′ end of the bHLH or PAS domains and the 3′ ends of the PAS domains. PCR products of appropriate sizes were obtained from both tissues for AHR (∼ 600 bp) and ARNT (∼ 1 kb). Sequence analysis revealed high sequence identity with AHR1 and ARNT1, respectively, of other species. The mRNA for the two transcription factors was easily detectable and levels were similar in adrenal and liver tissue of control animals. Following 3MC treatment, ARNT, but not AHR, mRNA increased to the same extent in both tissues. The similarity in control and induced expression of AHR and ARNT in the two tissues suggests that other, tissue-specific, factors are involved in the differential regulation of CYP1A1 and CYP1A2 in the adrenal vs liver. (Supported by NIH DK39671, DK07421, HL 48476, ES00260, and ES07381).

Chemical modifications of proteins, DNA and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests these RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N ⁶ -methyladenosine (m6A). In a subset of mRNAs, m ⁶ A sites are preferentially enriched near stop codons, in 3’ UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 hours starting from 72 hours post-fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Our analysis revealed 117 and 217 m6A peaks in the DMSO and PCB126 samples (FDR 5%), respectively. The majority of the peaks were preferentially located around the 3’UTR and stop codons. Statistical analysis revealed 15 m6A marked transcripts to be differentially methylated by PCB126 exposure. These include transcripts that are known to be activated by AHR agonists (e.g., ahrra, tiparp, nfe2l2b) as well as others that are important for normal development (vgf, cebpd, foxi1). These results suggest that environmental chemicals such as dioxin-like PCBs could affect developmental gene expression patterns by altering m6A levels. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels.