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RNAi High Throughput Screenings Facilitate the Identification of Proteins Necessary for TCDD-induced CYP1A1 Enzymatic Activity and Aid in the Discovery of a Novel Role for Sin3A in AHR-Mediated Gene Expression

  • Author(s): Solaimani, Parrisa Sherry
  • Advisor(s): Hankinson, Oliver
  • et al.
Abstract

The aryl hydrocarbon receptor (AHR) pathway is activated upon exposure to environmental pollutants 2,3,7,8-tetrachlorodibenzo-�-dioxin (TCDD) and benzo[a]pyrene (B[a]P), which leads to the induced expression of several genes. Toxicity of AHR occurs through the bioactivation of procarcinogens and eicosanoids by its target genes. In this study we identified several proteins that modulate the induction of the AHR target gene Cyp1a1. For this purpose we optimized procedures for the use of an RNAi high throughput screening, as well as for the construction and use of endoribonuclease-prepared siRNA (esiRNA) to validate the screening results. Expression of PDC, CD9, TMEM5, and Sin3A were found to be necessary for the induction of CYP1A1 whereas expression of Rbm5, ARMC8, PDC, CD9, TMEM5, Sin3A, Rab40C, Rad50, and Ube2i were necessary for both AHR expression and CYP1A1 induction. Additional studies were performed on the transcription factor candidate Sin3A, from which we found that Sin3A physically associates with the 5'-flanking regulatory regions of CYP1A1 in both human and mouse cell lines, and may potentially act as a coactivator for CYP1A1 induction. These studies established an essential role for Sin3A in the AHR-mediated induction of gene expression. We next examined the role of eicosanoids in AHR-dependent TCDD toxicity. CYP1A1, and other cytochrome P450s such as CYP2S1, can metabolize arachidonic acid into a variety of bioactive eicosanoids which play a significant role in the inflammatory response. From these studies we found that TCDD increased the levels of eicosanoids likely generated by cytochrome P450s in several tissues. Furthermore, these changes correlated with an increase in CYP1A1, CYP1B1, and CYP1A2 mRNA expression and were observed in wildtype mice but not AhR null mice. This demonstrated that these effects are mediated through AHR. Lastly, we explored dexamethasone-mediated regulation of CYP2S1. An initial screening looking for inhibitors of CYP2S1 revealed that dexamethasone, a glucocorticoid receptor (GR) ligand used to treat inflammatory diseases, represses its expression in multiple cell lines. We further found that dexamethasone regulates CYP2S1 via the GR and this occurs through the recruitment of histone deacetylases to the CYP2S1 promoter and enhancer.

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