title: Genome-wide RNAi high-throughput screen identifies proteins necessary for the AHR-dependent induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin.
abstract: The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes.
authors.affiliation: * Molecular Toxicology Interdepartmental Program, Department of Pathology and Laboratory Medicine, and the Jonsson Comprehensive Cancer Center and.
authors.forename: Parrisa
authors.initials: P
authors.lastname: Solaimani
authors.forename: Robert
authors.initials: R
authors.lastname: Damoiseaux
authors.forename: Oliver
authors.initials: O
authors.lastname: Hankinson
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D020555:GTP-Binding Protein Regulators / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D055106:Genome-Wide Association Study; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D057166:High-Throughput Screening Assays; D051379:Mice; D010078:Oxazines / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D013379:Substrate Specificity; D060245:Tetraspanin 29 / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D019363:Cytochrome P-450 CYP1A1; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D010078:Oxazines; D010750:Phosphoproteins; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D012097:Repressor Proteins; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D060245:Tetraspanin 29; C111235:AHR protein, human; C554870:Cd9 protein, mouse; C099683:SIN3A transcription factor; C065631:phosducin; C007649:ethoxyresorufin
keywords: AHR; CYP1A1; RNAi; TCDD; esiRNA.; high-throughput screening