A CRISPR-Cas9 screen for novel proteins required for induction of CYP1A1 by AHR

CRISPR-Cas9 筛选 AHR 诱导 CYP1A1 所需的新型蛋白质

• 批准号: 9276681
• 负责人: OLIVER nmn HANKINSON
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9276681
• 关键词: ARNT protein; Adverse effects; Affect; Agonist; Alleles; Aromatic Polycyclic Hydrocarbons; Aryl Hydrocarbon Receptor; Base Sequence; Benzo(a)pyrene; Binding; CRISPR library; CRISPR screen; CRISPR/Cas technology; CYP1A1 gene; CYP1A2 gene; CYP1B1 gene; Carcinogens; Cell Line; Cell Nucleus; Cells; Chemicals; Chromatin Structure; Code; Cultured Cells; DNA Double Strand Break; Dimerization; Dioxins; Diploid Cells; Enhancers; Environmental Pollutants; Exhibits; Family member; Foundations; Frameshift Mutation; Frequencies; Future; Gene Expression; Generations; Genes; Genetic Transcription; Genomics; Goals; Guide RNA; Hypoxia; Hypoxia Inducible Factor; Knockout Mice; Lead; Lentivirus Vector; Libraries; Ligands; Mammalian Cell; Mammals; Mediating; Messenger RNA; MicroRNAs; Mus; Play; Polychlorinated Biphenyls; Proteins; RNA Polymerase II; Recruitment Activity; Resistance; Role; Smog; System; Testing; Tetrachlorodibenzodioxin; Tissues; Tobacco smoke; Toxic effect; Transcriptional Activation; Validation; Variant; aryl hydrocarbon receptor ligand; carcinogenesis; carcinogenicity; cellular transduction; cytotoxic; dibenzofuran; dimer; gene induction; gene product; hepatoma cell; in vivo; interest; loss of function; mutant; novel; prevent; promoter; public health relevance; receptor binding; response; screening; tumor growth; tumor progression; vector

 DESCRIPTION (provided by applicant): 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.

 描述（申请人提供）：环境污染物2,3,7,8-四氯二苯并-对二恶英（TCDD）具有大量毒性作用，包括致癌性。 TCDD 和相关多氯化合物的所有毒性作用均由芳基烃受体 (AHR) 介导，并取决于 AHR 对尚未完全鉴定的下游基因的转录激活。 AHR 还介导多环芳烃（例如苯并[a]芘）的致癌作用，多环芳烃是烟草烟雾和烟雾中的重要致癌物。 CYP1A1、CYP1A2 和 CYP1B1 在许多组织中被大量诱导，并且可能通过将 PAH 代谢为亲电子衍生物来介导 PAH 的致癌作用。与激动剂结合后，AHR易位到细胞核并与芳基烃核易位蛋白（ARNT）形成二聚体，然后与反应基因的增强子区域结合，从而导致其转录激活。在当前的提案中，我们将寻求识别在以下方面发挥作用的新型基因产物： 通过筛选 CRISPR-Cas9 文库，AHR 诱导基因转录。 CRISPR-Cas9系统可以通过引导RNA（gRNA）内合成的20个核苷酸序列在特定基因组位点诱导DNA双链断裂，当靶向基因的编码区域时，可以产生缺失或插入，从而导致移码突变，导致二倍体细胞中两个等位基因的功能丧失。有两个具体目标。在具体目标 1 中， CYP1A1 诱导所需的基因将通过使用 GeCKOv2 文库进行鉴定，该文库针对 20,611 个小鼠基因（以及几乎所有蛋白质编码基因）和 1,178 个 microRNA。该文库包含在单个高效价慢病毒载体 (lentiCRISPRv2) 中， 将被转导至小鼠肝癌细胞系 Hepa-1 中。转导的细胞将在苯并[a]芘中选择10天。 （苯并[a]芘抗性克隆表现出 AHR 依赖性 CYP1A1 诱导的丧失）。然后对整合的 gRNA 进行 PCR 扩增及其测序。由超过一种 gRNA 代表的、和/或在超过一种转导的培养物中鉴定出的、和/或以高频率代表的那些基因可能代表真阳性。特定目标 2 将验证屏幕上的命中。每个感兴趣基因的两个 gRNA 将被插入 lentiCRISPRv2 中，以确定它们是否赋予苯并[a]芘抗性，减少 CYP1A1 的 TCDD 诱导，并在转导到 Hepa-1 细胞后减少 AHR 和 ARNT 表达。将在某些转导子中对相应的内源基因进行测序，以查看它们是否包含缺失或插入。已确认命中的特征将在未来的 R01 应用中进行。此类命中可能包括影响 AHR 或 ARNT 表达或功能和/或 CYP1A1 转录激活所需的基因产物。因此，当前的提议为全面分析 AHR 依赖性基因转录诱导机制奠定了基础，并可能导致减少 AHR 配体有害影响的策略。