The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a large number of toxic effects, including carcinogenicity. All the toxic effects of TCDD and of related polychlorinated compounds are mediated by the Aryl Hydrocarbon Receptor (AHR) and depend upon transcriptional activation by the AHR of not yet fully identified downstream genes. AHR also mediates carcinogenesis by polycyclic aromatic hydrocarbons, (e.g. benzo[a]pyrene) which are important carcinogens in tobacco smoke and smog. CYP1A1, CYP1A2 and CYP1B1 are massively induced in many tissues and are likely responsible for mediating the carcinogenic effects of PAHs, via metabolizing them to electrophilic derivatives. After binding agonist, the AHR translocates to the nucleus and forms a dimer with the aryl hydrocarbon nuclear translocator (ARNT) protein, which then binds to the enhancer regions of responsive genes, thereby leading to their transcriptional activation. In the current proposal we will seek to identify novel gene products that play roles in the induction of gene transcription by AHR, by screening a CRISPR-Cas9 library. The CRISPR-Cas9 system can induce DNA double strand breaks at specific genomic loci through synthetic 20 nucleotide sequences within guide RNAs (gRNAs), which when targeted to coding regions of genes can generation of deletions or insertions, resulting in frameshift mutations which lead to loss of function at both alleles in a diploid cell. There are two specific aims.
In Specific Aim 1, genes required for CYP1A1 induction will be identified by using the GeCKOv2 library, which targets 20,611 mouse genes (and thus nearly all protein coding genes) and also 1,178 microRNAs. The library is contained in a single high titer lentiviral vector (lentiCRISPRv2), which will be transduced into the mouse hepatoma cell line, Hepa-1. The transduced cells will be selected for 10 days in benzo[a]pyrene. (Benzo[a]pyrene-resistant clones exhibit loss of AHR-dependent induction of CYP1A1). PCR amplification of the integrated gRNAs and their sequencing will then be performed. Those genes represented by more than one gRNA, and/or identified in more than one transduced culture, and/or represented at high frequency, are likely to represent true positives.
Specific Aim 2 will validate hits from the screen. Two gRNAs for each gene of interest will be inserted into lentiCRISPRv2 to determine if they confer benzo[a]pyrene resistance, reduce TCDD induction of CYP1A1, and reduce AHR and ARNT expression upon transduction into Hepa-1 cells. The corresponding endogenous genes will be sequenced in certain transductants to see if they contain deletions or insertions. Characterization of confirmed hits will be pursued in a future R01 application. Such hits may include gene products that affect AHR or ARNT expression or function, and/or that are required for transcriptional activation of CYP1A1. The current proposal therefore lays the foundations for a comprehensive analysis of the mechanism of AHR-dependent induction of gene transcription, and may lead to strategies for reducing the harmful effects of ligands of the AHR.
The Aryl Hydrocarbon Receptor (AHR) binds and mediates the toxic effects of a wide variety of environmental pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related polychlorinated compounds, and polycyclic aromatic hydrocarbons (important carcinogenic components of tobacco smoke and smog) via stimulating the expression of a number of genes. A better understanding of how the AHR activates gene expression, which is the goal of this project, has the potential to generate strategies for preventing or ameliorating the toxic effects of the above chemicals. In order to achieve this objective we will screen a recently described CRISPR-Cas9 library targeting nearly all the coding genes in the mouse, and identify novel genes required for AHR-dependent induction of gene expression.
