The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor in the basic-helix-loophelix/Per-ARNT-Sim (bHLH-PAS) family. Halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) act through the AHR to cause altered expression of xenobiotic-metabolizing enzymes and other changes leading to toxicity in mammals, fish, and other vertebrate animals. However, the normal functions of the AHR and the exact mechanisms by which TCDD and related chemicals act through the AHR to cause toxicity are poorly understood. We and others have recently characterized three members of the vertebrate AHR family: AHR1, AHR2, and AHR Repressor (AHRR), and we have found that there is additional AHR diversity in fish, with up to 5 genes per species. We propose an integrated set of studies in vertebrate model systems (fish, mouse cells, human cells) that will take advantage of the unique features of each model to better understand the function of AHR and AHRR proteins and their roles in dioxin toxicity and normal development. 1) We will use an RNA knock-down strategy employing morpholino anti-sense technology in zebrafish (Danio rerio) embryos to test the hypothesis that AHR1, AHR2, and AHRR have distinct roles during development and in dioxin toxicity. These studies will take advantage of the external development and transparent embryos of zebrafish. The effect of blocking AHR 1, AHR2, and AHRR expression on sensitivity to dioxin developmental toxicity and gene expression will also be determined. 2) We will test the hypothesis that multiple AHRs from fish have undergone subfunctionalization and can therefore be used to distinguish multiple functions of the human AHR. Multiple AHRs from medaka (Oryzias latipes) and pufferfish (Fugu rubripes) will be characterized in vitro and expressed in AHR-deficient mouse cells ? TCDD to determine whether individual fish AHRs regulate distinct subsets of genes controlled by the murine AHR. Taking advantage of the compact genome of pufferfish, we will also conduct in vivo mapping of AHR promoters and regulatory elements using zebrafish embryos expressing GFP reporter constructs. 3) We will characterize the function, expression, and regulation of the human AHRR, examining inducibility by AHR agonists, structure-activity and dose-response relationships, and regulatory elements involved in AHRR induction. We will test the hypotheses that AHRR inhibition of AHR function occurs through competition for ARNT and/or AHR response elements. These studies will provide a better understanding of the possible role of human AHRR in modulating dioxin effects.
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