Worm Breeder's Gazette 14(5): 72 (February 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Molecular, Cellular and Developmental Biology, University of Colorado, Boulder C0 80309-0347
The vertebrate aryl hydrocarbon receptor (AHR; a.k.a. the dioxin receptor) mediates the teratogenic and carcinogenic effects of certain environmental contaminants. AHR ligands include benzo(a)pyrene, the primary carcinogen in cigarette smoke, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous man-made pollutant. Unliganded AHR resides in the cytoplasm in a complex with the 90-kDa heat shock protein (HSP90). Upon binding ligand, AHR translocates to the nucleus, dissociates from HSP90, and dimerizes with the AHR nuclear translocator protein (ARNT). Both AHR and ARNT belong to a class of regulatory proteins that contain a basic-helix-loop-helix DNA binding motif and a PAS domain (also found in Drosophila period and single-minded). The AHR-ARNT heterodimer binds specific promoter sequences to regulate transcription. (1) In mice AHR function is required for the proper development of the immune system and liver, but the presumptive endogenous ligand(s) is unknown. (2) Until recently, it had not been possible to study AHR signaling in a model system amenable to genetic analysis, as no invertebrate orthologues of AHR or ARNT had been reported. We have cloned the C. elegans homologues of AHR and ARNT. CeAHR may be the egl-33 gene; it is included in the 15kb genomic fragment that rescues egl-33 (ct315). (3) CeARNT maps to the right of egl-33 on LGI. We have expressed these gene products in rabbit reticulocyte lysates. Initial gel electrophoretic shift experiments demonstrate that CeAHR and CeARNT interact to bind the mammalian AHR-ARNT enhancer site. Competition experiments with wild-type and a mutant form of the enhancer site confirm that this binding is specific. These data suggest that we have cloned true C. elegans orthologues of AHR and ARNT. We are also collaborating with C. Bradfield and colleagues at the University of Wisconsin to determine the binding affinity of CeAHR for TCDD. To identify the cellular decisions regulated by the AHR signaling complex in C. elegans, we are screening for Tc1-induced deletions in CeAHR and CeARNT, and we are examining the expression patterns of these two genes. RNA blots indicate that CeAHR and CeARNT are coordinately expressed and are most abundant during embryogenesis and the first larval stage. We have constructed lacZ and GFP reporter genes and have detected expression of CeAHR::lacZ as early as the 30-cell stage. At the 300-cell stage, it is expressed in approximately 20% of the cells. As morphogenesis begins, most cells cease to express CeAHR::lacZ, and at hatching, it is detectable in a subset of neuronal and hypodermal cells. These experiments will establish the foundation for further genetic analysis of the AHR signaling pathway. 1. Hankinson, O. (1995) Annu. Rev. Pharmacol. Toxicol. 35: 307-34; Whitlock, J. P. (1993) Chem. Res. Toxicol. 6: 754-763; Bock, K. W. (1993) Rev. Phys. Biochem. Pharmacol. 125: 1-42 2. Fernandez-Salguero, P. et al. (1995) Science 268: 722-726 3. Powell-Coffman, J. A. and B. Wood. 1996 West Coast Worm Mtg.