Genomic profiling of yeast resistance to AFB1, a P450-activated carcinogen

酵母对 AFB1（一种 P450 激活的致癌物）抗性的基因组分析

基本信息

批准号：
8256182
负责人：
MICHAEL Thomas FASULLO
金额：
$ 6.2万
依托单位：
STATE UNIVERSITY OF NEW YORK AT ALBANY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-15 至 2014-01-14
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8256182
关键词：
Aflatoxin B1 Alleles Animal Model Bar Codes Benzo(a)pyrene Biological Assay Biological Models Bypass CHEK2 gene CYP1A1 gene CYP1A2 gene Carcinogen Resistance Carcinogens Cell physiology Cells Chemical Agents Chromosome Mapping Collaborations Collection Communities Computational Biology Computational Technique Computer Analysis Computing Methodologies Cytochrome P450 DNA DNA Adducts DNA Damage DNA Microarray Chip DNA Repair DNA Repair Gene DNA Repair Pathway DNA Sequence DNA biosynthesis DNA damage checkpoint DNA replication fork Data Diploidy Double Strand Break Repair Environmental Carcinogens Environmental Exposure Environmental Risk Factor Enzymes Epidemiologic Studies Food Gene Deletion Genes Genetic Genetic Epistasis Genetic Polymorphism Genetic Predisposition to Disease Genetic Recombination Genome Genomics Grant Growth Haploidy Heterocyclic Amines Housekeeping Gene Human Individual Knock-out Lesion Libraries Maintenance Malignant Neoplasms Mammals Measures Mediating Metabolic Activation Metabolism Methods Methyl Methanesulfonate Modeling Molecular Nucleotide Excision Repair Ontology Pathway interactions Patients Phenotype Plasmids Predisposition Process Publishing Radiation Research Research Personnel Resistance Resistance profile Resolution Resources Risk S Phase Saccharomyces cerevisiae Saccharomycetales Sample Size System Systems Biology Tissues Toxic effect Training Yeasts base cancer risk cell growth cohesion design environmental agent fitness gene function high throughput analysis high throughput screening insight man mutant novel recombinational repair repaired research study resistant strain response tool

项目摘要

DESCRIPTION (provided by applicant): Environmental factors are estimated to be the cause of 19% of cancers. However, human susceptibility to environmental carcinogens is highly variable. Low penetrant alleles may increase the risk for particular environmental-associated cancers. Epidemiological studies, however, are limited by their statistical power and the requirement for large patient numbers. Because many DNA metabolism and housekeeping genes are conserved from yeast to man, high throughput analysis of Saccharomyces cerevisiae (budding yeast) genes that confer resistance to carcinogens have identified human genes that confer resistance to environmental carcinogens. Genomic phenotyping using the ~5,000 yeast single-gene deletion haploid and diploid strains have been highly successful in determining genes that confer resistance to radiation and chemical agents. However, 75% of environmental agents are not carcinogenic per se, but require bioactivation, such as tissue - specific cytochrome P450-mediated metabolic activation. We previously were successful in introducing human CYP1A2 and CYP1A1 into yeast and activating a variety of carcinogens, including aflatoxin B1 (AFB1), benzo[a]pyrene dihydrodiol (BaP-DHD), and heterocyclic amines (HA, food carcinogens). The aim of this project is to determine which yeast genes are required for resistance to the potent carcinogen, AFB1, using a high throughput systems biology approach. We will introduce plasmids that express human CYP1A2 into the ~5,000 diploid homozygous single-deletion and heterozygous single-deletion strains. In the first aim, we will profile the yeat genome using the diploid single deletion strains for resistance to AFB1. Genes that confer resistance will be identified by high-throughput sensitive assays to measure cell growth and by molecular bar codes using high throughput sequencing or microarrays. Considering that AFB1 is a potent recombinagen, in the second aim, we will identify DNA repair and recombination pathways that confer AFB1 resistance by high throughput sensitivity analysis of double mutants derived from a set of single DNA repair mutants. These studies will thus identify novel genes involved in mediating carcinogen resistance and sensitivity, and provide insights into how recombinational repair processes actively tolerate DNA adducts. The strain collections will be made available to the scientific community and will be a valuable resource for characterizing the genetic susceptibility to environmental agents. The project will be a valuable training tool in systems and computational biology. PUBLIC HEALTH RELEVANCE: Sporadic cancer likely results from a combination of environmental exposure and genetics. Many genetic polymorphisms contribute to cancer risk. However, studies to identify these polymorphisms often lack statistical significance due to small sample sizes. High throughput screening in model organisms will facilitate identifying genes that confer resistance to environmental carcinogens. Since many DNA repair genes are conserved from yeast to mammals, high throughput screening has been successful in identifying genes that confer resistance to multiple DNA damaging agents. Many environmental carcinogens, however, must be metabolically activated by cytochrome P450 enzymes, which are lacking in yeast. We propose to introduce the human P450 gene CYP1A2 into the diploid yeast deletion collection. By high throughput screening we will define a genomic profile for resistance to aflatoxin B1. By computational methods, we will determine DNA repair pathways that confer aflatoxin B1 resistance. This project will give me valuable training in Systems Biology approaches, which will be a powerful method for genomic profiling of resistance to many environmental DNA damaging agents.

描述（由申请人提供）：估计环境因素是19％的癌症的原因。但是，人类对环境致癌物的敏感性是高度可变的。低渗透剂等位基因可能会增加特定环境相关癌症的风险。但是，流行病学研究受其统计能力和对大量患者人数的要求的限制。由于许多DNA代谢和管家基因都是从酵母到人的保存，因此对抗性毒素抗性的酿酒酵母（发芽酵母）基因的高吞吐量分析已经鉴定出赋予对环境癌症的耐药性的人类基因。使用约5,000个酵母单基因缺失单倍体和二倍体菌株的基因组表型在确定对辐射和化学剂抗性的基因方面非常成功。然而，75％的环境药物本身不是致癌的，而是需要生物活化，例如组织 - 特异性细胞色素P450介导的代谢激活。我们以前成功地将人CYP1A2和CYP1A1引入酵母中，并激活各种致癌物，包括黄霉素B1（AFB1），苯并[A] pyrene二氢二醇（BAP-DHD）和杂环胺（HA，食品营养物）。该项目的目的是确定使用高吞吐量系统生物学方法对有效致癌AFB1的抗性所需的酵母基因。我们将引入将人CYP1A2表达的质粒中，将约5,000个二倍体纯合单滴和杂合的单排除菌株中。在第一个目标中，我们将使用二倍体单个缺失菌株对YEAT基因组进行介绍，以抗AFB1。赋予电阻的基因将通过高通量敏感的测定方法来鉴定，以测量细胞生长以及使用高吞吐量测序或微阵列测量细胞生长和分子条形码。考虑到AFB1是一种有效的重组，在第二个目标中，我们将确定DNA修复和重组途径，这些途径通过一组单个DNA修复突变体得出的双突变体对双突变体的高吞吐量敏感性分析赋予AFB1抗性。因此，这些研究将确定参与介导致癌性和敏感性的新型基因，并提供有关重组修复过程如何积极耐受DNA加合物的见解。应变收集将提供给科学界，将是表征对环境药物的遗传敏感性的宝贵资源。该项目将是系统和计算生物学方面的宝贵培训工具。公共卫生相关性：零星癌可能是由于环境暴露和遗传学的结合而引起的。许多遗传多态性有助于癌症风险。但是，确定这些多态性的研究通常由于样本量较小而缺乏统计学意义。模型生物中的高吞吐量筛选将有助于识别赋予环境致癌物的耐药性的基因。由于许多DNA修复基因从酵母到哺乳动物都是保守的，因此高吞吐量筛选成功地鉴定了赋予多种DNA损伤剂的耐药性的基因。然而，许多环境致癌物必须通过酵母中缺乏的细胞色素P450酶来代谢。我们建议将人P450基因CYP1A2引入二倍体酵母缺失收集。通过高吞吐量筛选，我们将定义一个基因组谱，以抗性黄曲霉毒素B1。通过计算方法，我们将确定赋予黄曲霉毒素B1抗性的DNA修复途径。该项目将为我提供系统生物学方法的宝贵培训，这将是对许多环境DNA损害剂的抗性基因组分析的有力方法。