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

基本信息

批准号：
9112338
负责人：
OLIVER nmn HANKINSON
金额：
$ 19.25万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9112338
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 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 Tissues Tobacco smoke Toxic effect Transcriptional Activation Validation Variant aryl hydrocarbon receptor ligand carcinogenesis carcinogenicity cellular transduction cytotoxic dibenzofuran dimer gene product hepatoma cell in vivo interest loss of function mutant novel prevent promoter public health relevance response screening tumor growth tumor progression vector

项目摘要

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（由适用提供）：环境污染物2,3,7,8-四氯迪本佐-P-二恶英（TCDD）具有大量的毒性作用，包括致癌性。 TCDD和相关多氯化化合物的所有毒性作用均由芳基烃受体（AHR）介导，并依赖于尚未完全鉴定的下游基因的AHR转录激活。 AHR还通过多环芳族碳氢化合物（例如苯并[a] pyrene）介导了癌变，它们是烟草烟雾和烟雾中重要的致癌物。 CYP1A1，CYP1A2和CYP1B1在许多组织中被大量诱导，并且可能是通过将它们代谢为亲电衍生物来介导PAHS的致癌作用的原因。结合激动剂后，AHR转移到核并用芳基烃核转运剂（ARNT）蛋白形成二聚体，然后与响应基因的增强子区域结合，从而导致其转录激活。在当前的建议中，我们将寻求确定在中发挥作用的新型基因产品 CRISPR-CAS9系统可以通过指导RNA（GRNA）内的合成20个核苷酸序列在特定的基因组基因座处诱导DNA双链断裂，当该核苷酸（GRNA）靶向基因的编码区域时，可以产生缺失或插入的编码区域，从而导致在偶发性细胞中两个异地的功能丧失的帧速率突变。有两个具体的目标。在特定的目标1中， CYP1A1诱导所需的基因将通过使用Geckov2库来鉴定，该库针对20,611个小鼠基因（因此几乎所有蛋白质编码基因）以及1,178个microRNA。该库中包含在单个高滴度慢病毒载体（Lenticrisprv2）中，该库中包含在该库中转导的细胞将在苯并[a] pyrene中选择10天。（苯并[A]抗逆性克隆暴露于CYP1A1的AHR依赖性诱导损失）。然后将进行集成GRNA及其测序的PCR扩增。那些由多个GRNA代表的基因和/或在一个以上转导的培养物中被鉴定出来的，并且/或以高频表示，可能代表真实的阳性。特定的AIM 2将验证屏幕上的命中。每个感兴趣的基因的两个GRNA将插入Lenticrisprv2中，以确定它们是否赋予苯并[A] pyrene耐药性，降低TCDD的CYP1A1诱导，并在转移到HEPA-1细胞中时降低AHR和ARNT的表达。相应的内源基因将在某些转导剂中进行测序，以查看它们是否包含缺失或插入。将来的R01应用程序将进行确认的命中的表征。此类命中可能包括影响AHR或ARNT表达或功能的基因产物，以及/或CYP1A1的转录激活所需的基因产物。因此，当前的建议为基础奠定了对基因转录依赖AHR依赖性诱导机制的全面分析的基础，并可能导致降低AHR配体的有害作用的策略。