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.

Selective Inhibition of FOXO1 Activator/Repressor Balance Modulates Hepatic Glucose Handling

“…Interestingly, cholesterol content is also decreased in iL -Sin3a/b mice. This can reflect GCK- or/and FOXO1-independent functions of SIN3A in liver metabolism, for example through its role to regulate mitochondrial activity (Barnes et al, 2014; Pile et al, 2003), or cholesterol biosynthesis (Solaimani et al, 2013). There are likely to be additional functions to hepatic SIN3A, in particular in the development of bile ducts, that account for the abnormal liver function tests, histopathologic abnormalities, and splenomegaly (likely secondary to portal hypertension) seen in mice with constitutive liver deletion.…”

SIN3A, Generally Regarded as a Transcriptional Repressor, Is Required for Induction of Gene Transcription by the Aryl Hydrocarbon Receptor

“…However, these siRNAs did not significantly reduce induction of CYP1A1 EROD activity (19). It is possible that these subunits are not essential for CYP1A1 induction.…”

“…Understanding the regulation of CYP1A1 will therefore contribute not only to an understanding of the dysregulation of AHR target genes but also the resulting toxicity of this dysregulation. We previously reported results from an RNAi high throughput screening we performed that indicated that SIN3A is necessary for TCDD induction of CYP1A1 in murine Hepa-1 cells (19). In this paper, we also demonstrate that overexpression of human SIN3A in Hepa-1 cells transfected with siRNA to mouse SIN3A was sufficient to rescue CYP1A1-mediated EROD activity, which validates our previous results and provides convincing evidence that our observations are a result of effects of the siRNA on SIN3A and not due to off-target effects.…”

“…Hepatoma Cell Line, Hepa-1-We previously performed an RNAi high throughput screening to identify proteins necessary for induction of CYP1A1-dependent EROD enzymatic activity (19). In this screening, four individual siRNAs per target gene were tested in duplicate, and each candidate gene was then ranked according to both the quality of the assay and the quantity of hits.…”

“…In a recent study, we aimed to further elucidate the mechanism leading to induction of CYP1A1 by performing an RNAi high throughput screening (19). For these studies, we used the mouse hepatoma cell line Hepa1c1c7 (henceforth called Hepa-1), in which CYP1A1 mRNA is induced many hundred-fold within 24 h of treatment with TCDD (1).…”