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National Institute of Environmental Health Sciences

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Your Environment. Your Health.

Publication Detail

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.

Authors: Solaimani, Parrisa; Damoiseaux, Robert; Hankinson, Oliver

Published In Toxicol Sci, (2013 Nov)

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.

PubMed ID: 23997114 Exiting the NIEHS site

MeSH Terms: Animals; Basic Helix-Loop-Helix Transcription Factors/agonists*; Basic Helix-Loop-Helix Transcription Factors/genetics; Basic Helix-Loop-Helix Transcription Factors/metabolism; Cell Line, Tumor; Cytochrome P-450 CYP1A1/biosynthesis*; Cytochrome P-450 CYP1A1/genetics; Enzyme Induction; Eye Proteins/genetics; Eye Proteins/metabolism; GTP-Binding Protein Regulators/genetics; GTP-Binding Protein Regulators/metabolism; Genes, Reporter; Genome-Wide Association Study; Hepatocytes/drug effects*; Hepatocytes/enzymology; High-Throughput Screening Assays*; Mice; Oxazines/metabolism; Phosphoproteins/genetics; Phosphoproteins/metabolism; Polychlorinated Dibenzodioxins/toxicity*; Polymerase Chain Reaction; RNA Interference*; RNA, Messenger/biosynthesis; Receptors, Aryl Hydrocarbon/agonists*; Receptors, Aryl Hydrocarbon/genetics; Receptors, Aryl Hydrocarbon/metabolism; Repressor Proteins/genetics; Repressor Proteins/metabolism; Reproducibility of Results; Substrate Specificity; Tetraspanin 29/genetics; Tetraspanin 29/metabolism; Transcription, Genetic/drug effects; Transfection

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