Matthew J. Jenny

Repression of Aryl Hydrocarbon Receptor (AHR) Signaling by AHR Repressor: Role of DNA Binding and Competition for AHR Nuclear Translocator

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin causes altered gene expression and toxicity. The AHR repressor (AHRR) inhibits AHR signaling through a proposed mechanism involving competition with AHR for dimerization with AHR nuclear translocator (ARNT) and binding to AHR-responsive enhancer elements (AHREs). We sought to delineate the relative roles of competition for ARNT and AHREs in the mechanism of repression. In transient transfections in which AHR2-dependent transactivation was repressed by AHRR1 or AHRR2, increasing ARNT expression failed to reverse the repression, suggesting that AHRR inhibition of AHR signaling does not occur through sequestration of ARNT. An AHRR1 point mutant (AHRR1-Y9F) that could not bind to AHREs but that retained its nuclear localization was only slightly reduced in its ability to repress AHR2, demonstrating that AHRR repression does not occur solely through competition for AHREs. When both proposed mechanisms were blocked (AHRR1-Y9F plus excess ARNT), AHRR remained functional. AHRR1 neither blocked AHR nuclear translocation nor reduced the levels of AHR2 protein. Experiments using AHRR1 C-terminal deletion mutants showed that amino acids 270 to 550 are dispensable for repression. These results demonstrate that repression of AHR transactivation by AHRR involves the N-terminal portion of AHRR; does not involve competition for ARNT; and does not require binding to AHREs, although AHRE binding can contribute to the repression. We propose a mechanism of AHRR action involving “transrepression” of AHR signaling through protein-protein interactions rather than by inhibition of the formation or DNA binding of the AHR-ARNT complex.

Potential Indicators of Stress Response Identified by Expressed Sequence Tag Analysis of Hemocytes and Embryos from the American Oyster, Crassostrea virginica

Abstract: A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: a tool for studying molecular mechanisms in an established environmental model.

A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in beta-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.

The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) binds multiple AHRs and induces multiple CYP1 genes via AHR2 in zebrafish

The tryptophan photooxidation product 6-formylindolo[3,2-b]carbazole (FICZ) has been proposed as a physiological ligand for the mammalian aryl hydrocarbon receptor (AHR), which it binds with high-affinity, inducing expression of cytochrome P450 1A1 (CYP1A1). We investigated whether the response to FICZ is evolutionarily conserved in vertebrates by measuring FICZ binding to two zebrafish AHRs (AHR1B and AHR2) and its ability to induce zebrafish CYP1 genes (CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1) in vivo. Exposure of zebrafish embryos (48 h-post-fertilization; hpf) to 10 nM FICZ for 6h caused strong induction of CYP1A mRNA and a statistically significant but modest induction of CYP1B1 and CYP1C1. Neither CYP1C2 nor CYP1D1 expression was induced by FICZ under the conditions of dose, time or developmental stage examined here. CYP1A induction was significantly greater after 6 h than after 12 h of exposure to FICZ, suggesting a rapid degradation of inducer. The 6-h EC(50) values for induction of CYP1A and CYP1B1 by FICZ were 0.6 and 0.5 nM compared to 72-h EC(50) values of 2.3 and 2.7 nM for PCB126, indicating that in zebrafish embryos FICZ is a more potent inducer than PCB126. FICZ at 10 nM was able to completely displace binding of 2,3,7,8-tetrachloro-1,6[3H]-dibenzo-p-dioxin to in vitro-expressed zebrafish AHR2 and AHR1B. Inhibition of AHR2 translation in zebrafish embryos by an AHR2-specific morpholino antisense oligonucleotide decreased the induction of CYP1A and CYP1B1 by FICZ and by PCB126. Together, these results demonstrate that FICZ is a potent AHR agonist in zebrafish, inducing expression of multiple CYP1 genes largely through AHR2. Evolutionary conservation of the response to FICZ is consistent with a possible role as an endogenous signaling molecule acting through the AHR.

Nrf2b, Novel Zebrafish Paralog of Oxidant-responsive Transcription Factor NF-E2-related Factor 2 (NRF2)

Background: NRF2 is a transcription factor that regulates the oxidative stress response. Results: Zebrafish have duplicate nrf2 genes, nrf2a and nrf2b, with distinct functions during embryonic development. Conclusion: nrf2a and nrf2b have undergone subfunction partitioning; Nrf2b is a negative regulator of embryonic gene expression. Significance: Duplicate zebrafish nrf2 genes provide opportunities for new insights into developmental roles of NRF2. NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.

Distinct Roles of Two Zebrafish AHR Repressors (AHRRa and AHRRb) in Embryonic Development and Regulating the Response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to dimethyl sulfoxide (DMSO) displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.

Marine Genomics: A clearing-house for genomic and transcriptomic data of marine organisms

BackgroundThe Marine Genomics project is a functional genomics initiative developed to provide a pipeline for the curation of Expressed Sequence Tags (ESTs) and gene expression microarray data for marine organisms. It provides a unique clearing-house for marine specific EST and microarray data and is currently available at http://www.marinegenomics.org.DescriptionThe Marine Genomics pipeline automates the processing, maintenance, storage and analysis of EST and microarray data for an increasing number of marine species. It currently contains 19 species databases (over 46,000 EST sequences) that are maintained by registered users from local and remote locations in Europe and South America in addition to the USA. A collection of analysis tools are implemented. These include a pipeline upload tool for EST FASTA file, sequence trace file and microarray data, an annotative text search, automated sequence trimming, sequence quality control (QA/QC) editing, sequence BLAST capabilities and a tool for interactive submission to GenBank. Another feature of this resource is the integration with a scientific computing analysis environment implemented by MATLAB.ConclusionThe conglomeration of multiple marine organisms with integrated analysis tools enables users to focus on the comprehensive descriptions of transcriptomic responses to typical marine stresses. This cross species data comparison and integration enables users to contain their research within a marine-oriented data management and analysis environment.

The Active Form of Human Aryl Hydrocarbon Receptor (AHR) Repressor Lacks Exon 8, and Its Pro185 and Ala185 Variants Repress both AHR and Hypoxia-Inducible Factor

ABSTRACT The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has been associated with reproductive dysfunction and tumorigenesis in humans. Previous studies have characterized the repressor function of AHRRs from mice and fish, but the human AHRR ortholog (AHRR715) appeared to be nonfunctional in vitro. Here, we report a novel human AHRR cDNA (AHRRΔ8) that lacks exon 8 of AHRR715. AHRRΔ8 was the predominant AHRR form expressed in human tissues and cell lines. AHRRΔ8 effectively repressed AHR-dependent transactivation, whereas AHRR715 was much less active. Similarly, AHRRΔ8, but not AHRR715, formed a complex with AHR nuclear translocator (ARNT). Repression of AHR by AHRRΔ8 was not relieved by overexpression of ARNT or AHR coactivators, suggesting that competition for these cofactors is not the mechanism of repression. AHRRΔ8 interacted weakly with AHR but did not inhibit its nuclear translocation. In a survey of transcription factor specificity, AHRRΔ8 did not repress the nuclear receptor pregnane X receptor or estrogen receptor α but did repress hypoxia-inducible factor (HIF)-dependent signaling. AHRRΔ8-Pro185 and -Ala185 variants, which have been linked to human reproductive disorders, both were capable of repressing AHR or HIF. Together, these results identify AHRRΔ8 as the active form of human AHRR and reveal novel aspects of its function and specificity as a repressor.

The Transcriptional Response to Oxidative Stress during Vertebrate Development: Effects of tert-Butylhydroquinone and 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.