Superfund Research Program
Mechanisms and Impacts of PCB Resistant Fish
Project Leader: Mark E. Hahn (Woods Hole Oceanographic Institution)
Co-Investigators: Sibel I. Karchner (Woods Hole Oceanographic Institution), Neelakanteswar Aluru (Woods Hole Oceanographic Institution)
Grant Number: P42ES007381
Funding Period: 1995-2020
Dr. Mark Hahn and his research team are studying a population of estuarine fish, Atlantic killifish (Fundulus heteroclitus), that have evolved resistance to polychlorinated biphenyls (PCBs). Killifish inhabiting New Bedford Harbor (NBH), MA, a PCB-contaminated Superfund site, are much less sensitive to PCBs than killifish from a reference site, Scorton Creek (SC). PCBs cause toxicity by activating aryl hydrocarbon receptor (AHR) proteins and killifish have at least two AHRs (AHR1 and AHR2). The objectives of the research are to determine the role of AHRs in PCB resistance and to evaluate the consequences of the PCB-resistant phenotype.
To determine whether the resistance of NBH fish to PCBs involves a limited number of AHR target genes or is more extensive and to generate information on the Fundulus transcriptome, the research group performed deep sequencing using 454/Life Sciences technology on 3’-anchored cDNA libraries from SC and NBH embryos that had been exposed to vehicle or 3,3’,4,4’,5-pentachlorobiphenyl (PCB-126) and sampled at 10 dpf. The libraries were sequenced in a half-run (Titanium FLX) from which they obtained ~634,000 reads. In addition, researchers performed shotgun sequencing of pooled polyA+ RNA from 1-15 dpf embryos (sampled every day) from SC and NBH. The two libraries were sequenced in a full-run (Titanium FLX), yielding ~1.5 million reads (750,000 reads per site). Together, these sequences facilitated the annotation of existing Fundulus cDNA clones and identified sequences of novel killifish genes that may be involved in the mechanism of PCB resistance in NBH fish.
To test the hypothesis that epigenetic changes are involved in the mechanism of resistance, the researchers cloned and sequenced AHR1 and AHR2 gene promoters and compared the methylation status of CpG islands of AHR1 and AHR2 in livers of adult killifish collected from NBH and SC. No significant differences in methylation profiles were observed between NBH and SC fish, for either gene. However, hypermethylation of the AHR1 promoter correlated with low expression of transcripts in the liver in both populations, suggesting that promoter methylation may control tissue-specific expression of AHR genes in killifish.
To further develop the killifish model and to begin to test hypotheses about the relative roles of AHR1 and AHR2, the research group initiated the generation of AHR2-null killifish using the new zinc-finger nuclease (ZFN) approach for targeted gene inactivation. They ordered custom-designed ZFNs targeting killifish AHR2. They injected capped mRNA encoding ZFNs into 1-cell killifish embryos (from SC). At 5 days post-fertilization, subsets of embryos were sampled from each group for analysis of mutations by DNA sequencing; the remaining 271 embryos are being raised to adulthood, after which they will be screened to identify founders (those carrying germ-line null mutations in AHR2).
The existence of dioxin-sensitive and dioxin-resistant populations of killifish provides a unique opportunity to understand the molecular mechanisms of differential dioxin sensitivity and the impact of evolved resistance on the sensitivity of fish to other environmental stressors. The focus this year on applying cutting-edge molecular approaches (454 pyrosequencing, epigenetics, ZFN gene targeting) will enhance the utility of the killifish model for addressing this and other mechanistic questions.