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

• 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
• 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

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）：据估计，19% 的癌症是由环境因素引起的。然而，人类对环境致癌物的易感性差异很大。低渗透等位基因可能会增加特定环境相关癌症的风险。然而，流行病学研究因其统计能力和需要大量患者而受到限制。由于许多 DNA 代谢和管家基因从酵母到人类都是保守的，因此对赋予致癌物抗性的酿酒酵母（芽殖酵母）基因进行高通量分析，已经鉴定出赋予对环境致癌物抗性的人类基因。使用约 5,000 个酵母单基因缺失单倍体和二倍体菌株进行基因组表型分析，在确定赋予辐射和化学试剂抗性的基因方面取得了巨大成功。然而，75%的环境因素本身并不致癌，但需要生物激活，例如组织特异性细胞色素P450介导的代谢激活。我们之前成功地将人类CYP1A2和CYP1A1引入酵母中并激活多种致癌物，包括黄曲霉毒素B1（AFB1）、苯并[a]芘二氢二醇（BaP-DHD）和杂环胺（HA，食品致癌物）。该项目的目的是使用高通量系统生物学方法确定哪些酵母基因是抵抗强致癌物 AFB1 所必需的。我们将把表达人 CYP1A2 的质粒引入约 5,000 个二倍体纯合单缺失和杂合单缺失菌株中。第一个目标是，我们将使用二倍体单缺失菌株来分析酵母基因组，以抵抗 AFB1。赋予抗性的基因将通过高通量敏感测定来鉴定以测量细胞生长，并通过使用高通量测序或微阵列的分子条形码来鉴定。考虑到 AFB1 是一种有效的重组原，在第二个目标中，我们将通过对一组单 DNA 修复突变体衍生的双突变体进行高通量灵敏度分析来确定赋予 AFB1 抗性的 DNA 修复和重组途径。因此，这些研究将鉴定出参与介导致癌物抗性和敏感性的新基因，并深入了解重组修复过程如何主动耐受 DNA 加合物。这些菌株收藏品将提供给科学界，并将成为表征对环境因素的遗传易感性的宝贵资源。该项目将成为系统和计算生物学方面的宝贵培训工具。 公共卫生相关性：散发性癌症可能是环境暴露和遗传因素共同作用的结果。许多基因多态性会增加癌症风险。然而，由于样本量较小，识别这些多态性的研究通常缺乏统计学意义。模式生物中的高通量筛选将有助于识别对环境致癌物具有抗性的基因。由于许多 DNA 修复基因从酵母到哺乳动物都是保守的，因此高通量筛选已成功鉴定出对多种 DNA 损伤剂具有抗性的基因。然而，许多环境致癌物必须通过细胞色素 P450 酶进行代谢激活，而酵母中缺乏这种酶。我们建议将人类 P450 基因 CYP1A2 引入二倍体酵母缺失集合中。通过高通量筛选，我们将确定黄曲霉毒素 B1 抗性的基因组谱。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。这个项目将为我提供系统生物学方法方面的宝贵培训，这将是对许多环境 DNA 损伤剂的抗性进行基因组分析的强大方法。