Department of Pathology and Laboratory Medicine, and Molecular Toxicology Program, University of California Los Angeles, Los Angeles, California 90095.
Toxicol Sci. 2019 Aug 1;170(2):310-319. doi: 10.1093/toxsci/kfz111.
Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.
环境污染物包括卤代芳烃和多环芳烃，如苯并[a]芘，通过激活芳烃受体（AHR）并转录尚未完全确定的基因发挥其有害作用。配体结合的 AHR 从细胞质易位到细胞核，在那里它与芳烃受体核转位蛋白（ARNT）蛋白二聚化。AHR/ARNT 二聚体结合到反应基因的增强子区域以激活转录。AHR 还介导多环芳烃引起的致癌作用，可能通过 CYP1A1、CYP1A2 和 CYP1B1，这些基因在许多组织中被激活的 AHR 大量诱导，并产生多环芳烃的致癌亲电衍生物。在本研究中，我们利用 B[a]P 选择测定法，利用我们之前用于鉴定 Hepa-1c1c7 小鼠肝癌细胞中核心 AHR 途径基因的测定法，利用小鼠 GeCKOv2 全基因组 CRISPR/Cas9 文库来鉴定 AHR 途径中的新基因。除了 Ahr、Arnt 和 Cyp1a1，我们还报告了多个额外的推定 AHR 途径基因的鉴定，包括我们验证的几个基因。这些基因包括细胞色素 P450 还原酶（Por），它介导细胞色素 P450 的氧化还原再生，以及血红素生物合成途径的 5 个基因：δ-氨基酮戊酸合酶 1（Alas1）、卟啉原脱氨酶（Hmbs）、尿卟啉原脱羧酶（Urod）、粪卟啉原氧化酶（Cpox）和亚铁螯合酶（Fech）：血红素是细胞色素 P450 蛋白的必需辅基。值得注意的是，尽管这些基因不能通过反向遗传学方法鉴定，但其中几个基因是通过 GeCKO 筛选鉴定的。这表明高通量全基因组遗传筛选在鉴定 AHR 途径中的基因方面具有强大的作用。
