Characterization of the aryl hydrocarbon receptor repressor in zebrafish, Danio rerio

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the aryl hydrocarbon receptor (AHR). Upon activation, the AHR forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT) and alters gene expression by binding AHR regulatory elements (AHRE). The AHR repressor (AHRR) forms a negative feedback loop with the AHR. This work describes the identification and functional analysis of AHRRs in the zebrafish, Danio rerio.; Two zebrafish AHRR cDNAs, AHRR1 and AHRR2, were cloned and sequenced. These are co-orthologs of the mammalian AHRR, as suggested by gene mapping and phylogenetic analyses. In transient transfection assays, AHRR1 and AHRR2 repressed AHR2-dependent constitutive and TCDD-inducible expression of an AHRE-regulated reporter gene. In ZF-L cells, both AHRRs were induced with structure-activity and dose-response relationships consistent with an AHR-dependent mechanism. Both repressors also were expressed and induced by TCDD in zebrafish embryos.; The mechanism of repression by AHRR was investigated. Overexpression of ARNT2 failed to prevent repression of AHR2 transcriptional activity by AHRRs. Similarly, AHRR1-Y9F, possessing a point mutation that disrupts AHRE binding, failed to reverse repression of AHR2. Over-expression of ARNT2 and co-transfection of AHRRI-Y9F also did not prevent repression. Thus, AHRR1 inhibition of AHR signaling does not occur solely through competition for ARNT2 or AHREs; a third mechanism must exist. Experiments using deletion mutants of AHRR1 showed that the C-terminal 362 amino acids of AHRR1 are not necessary for repression.; The role of AHRR1 in regulating CYP1A expression in zebrafish embryos was investigated. Morpholino oligonucleotides were designed to the AHRR1 translation initiation site (RRint) and splice sites (RR2eiss and RRie4ss). RRint blocked in vitro translation of AHRR1, but when injected into embryos, CYPIA expression or inducibility was not altered consistently. Neither RR2eiss nor RRie4ss reduced the levels of wild-type AHRR1 transcript in vivo. Therefore, the role of AHRR1 in developing zebrafish embryos remains unclear.; This research has identified two zebrafish AHRR paralogs that are expressed and inducible by an AHR-dependent mechanism. These AHRRs inhibit AHR signaling through a complex mechanism that is independent of DNA binding and does not involve competition for ARNT.