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.

Keywords: AHR; CYP1A1; RNAi; TCDD; esiRNA.; high-throughput screening.

Fig. 1.

RNAi high-throughput screening methods. Methods used for the high-throughput screening are summarized in this flowchart. Abbreviations: EROD, ethoxyresorufin-o-deethylase; RNAi, RNA interference; TCDD, 2,3,7,8-tetrachlorodibenzo-р-dioxin.

Fig. 2.

Transcript levels of CYP1A1, siRNA targets, ALDH3A1, and NQO1 after siRNA-mediated knockdown and TCDD induction. The most efficient siRNA of the 4 used in the high-throughput screening was selected to use for future follow-up experiments. Effects each siRNA had on induced CYP1A1 mRNA expression (a), and efficiency of knockdown by individual siRNAs (b) was assessed by quantifying each corresponding targeted transcript. Effects each siRNA had on induced ALDH3A1 (c), and NQO1 (d) mRNA expression was also determined. Transcripts were measured using SYBR Green real-time PCR, and each sample was normalized to the constitutively expressed glycolytic GAPDH gene. All results shown on (a) and (b) are statistically significant with a p value ≤ .005 relative to mock and scrambled (SCR) negative controls. SCR refers to a scrambled siRNA negative control. *p < .05, **p < .01, and ***p < .001 compared with mock + SCR controls. Abbreviations: mRNA, messenger RNA; TCDD, 2,3,7,8-tetrachlorodibenzo-р-dioxin.

Fig. 3.

EROD rescue methods. Hepa-1 cells were transduced with a retroviral pMSCV-ires/GFP vector expressing a human cDNA of interest, and GFP positive cells were isolated using fluorescent-activated cell sorting (FACS). Isolated cells were then transfected with siRNA directed at the mouse cDNA counterpart, treated with TCDD, and assayed for CYP1A1 EROD activity to see if the effects of the siRNA were rescued by overexpressing the cDNA. Abbreviations: cDNA, complementary DNA; EROD, ethoxyresorufin-o-deethylase; TCDD, 2,3,7,8-tetrachlorodibenzo-р-dioxin.

Fig. 4.

Overexpresing AHR rescues EROD activity in Hepa-1 cells treated with certain siRNAs. Hepa-1 cells were transduced with a pMSCV-ires/GFP retroviral expression vector containing human cDNA to overexpress AHR, treated with siRNAs to the individual mouse genes, and subsequently treated with 10nM TCDD. CYP1A1 EROD activity was measured to determine if overexpression was enough to rescue CYP1A1 EROD activity in the presence of each siRNA (SCR refers to a scrambled siRNA negative control). *p < .05, **p < .01, and ***p < .001 compared with empty vector control. Abbreviations: AHR, aryl hydrocarbon receptor; cDNA, complementary DNA; EROD, ethoxyresorufin-o-deethylase; RFU, relative fluorescence units; TCDD, 2,3,7,8-tetrachlorodibenzo-р-dioxin.

Fig. 5.

ARMC8, SIN3A, and TMEM5 are necessary for TCDD-induced CYP1A1 protein expression but not for AHR protein expression. a, Western blots of CYP1A1 and AHR were performed after transfection of Hepa-1 cells with siRNAs to the indicated genes in the presence of DMSO (−) or TCDD (+) (SCR refers to a scrambled siRNA negative control). b, Densitometry quantification of Western blots (normalized to GAPDH) revealed the need for ARMC8, SIN3A, and TMEM5 in CYP1A1 but not AHR protein expression. Abbreviations: AHR, aryl hydrocarbon receptor; DMSO, dimethyl sulfoxide; TCDD, 2,3,7,8-tetrachlorodibenzo-р-dioxin.

Fig. 6.

RAB40C and CRYGD are necessary for TCDD-induced CYP1A1 protein expression and AHR protein expression. a, Western blots of CYP1A1 and AHR were performed after transfection of Hepa-1 cells with siRNAs to the indicated genes in the presence of DMSO (−) or TCDD (+) (SCR refers to a scrambled siRNA negative control). b, Densitometry quantification of Western blots (normalized to GAPDH) revealed the need for RAB40C and CRYGD in both CYP1A1 and AHR protein expression. Abbreviations: AHR, aryl hydrocarbon receptor; DMSO, dimethyl sulfoxide; TCDD, 2,3,7,8-tetrachlorodibenzo-р-dioxin.

Fig. 7.

Induction of luciferase activity by the pGL-CYP1A1 reporter construct in the presence of siRNAs to the individual genes of interest. Luciferase activity was measured in Hepa-1 cells that were cotransfected with pGL-CYP1A1 and the indicated siRNAs to investigate the potential effects of the corresponding gene products of the CYP1A1 promoter. Results revealed a significant influence of each siRNA on luciferase activity, with a most noticeable decrease for ARMC8 and CD9 compared with the untreated control. In the case of TCF20, there was a substantial increase in luciferase activity. Data are representative of 3 experiments and all results shown are statistically significant with a p value ≤ .001 relative to pGL-1A1 negative controls. Abbreviation: RLU, relative luminescence units.

Fig. 8.

Ectopic expression of ARMC8 and RBM5 rescues CYP1A1 EROD activity. Hepa-1 cells were transduced with a pMSCV-ires/GFP retroviral expression vectors containing human cDNAs for ARMC8 (a) and RBM5 (b). Cells were subsequently treated with the corresponding siRNA followed by 10nM TCDD, and CYP1A1 EROD activity then was measured to determine if overexpression was enough to rescue CYP1A1 EROD activity in the presence of siRNA. The siRNAs to CYP1A1 and AHR served as positive controls. SCR refers to a scrambled siRNA negative control. *p < .05, **p < .01, and ***p < .001 compared with empty vector control or compared with the mock and SCR controls combined. Abbreviations: AHR, aryl hydrocarbon receptor; cDNA, complementary DNA; EROD, ethoxyresorufin-o-deethylase; RFU, relative fluorescence units; TCDD, 2,3,7,8-tetrachlorodibenzo-р-dioxin.