Functional Genomics of LINE-1 Retrotransposition

LINE-1 逆转录转座的功能基因组学

基本信息

批准号：
10612492
负责人：
Kenneth S. Ramos
金额：
$ 64.39万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-21 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612492
关键词：
Aromatic Polycyclic Hydrocarbons Automobile Driving Benzo(a)pyrene Binding Biological Biological Assay Biological Markers CRISPR screen Cancer cell line Cancerous Carcinogens Cell Line Cell Respiration Cells Chromatin Structure Cities Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA DNA Adduction DNA Adducts DNA Damage DNA Methylation DNA sequencing Development Disease Disparity Elements Environment Environmental Carcinogens Environmental Pollution Epigenetic Process Epithelial Cells Event Exposure to Family Female Genes Genetic Genetic Polymorphism Genetic Predisposition to Disease Genetic Recombination Genetic study Genome Genomic Instability Heterogeneity Human Human Genome Hydrocarbons In Vitro Instruction Investigation KRAS2 gene Knowledge Laboratories Length Life Cycle Stages Light Link Liver Location Long Interspersed Elements Lung Malignant - descriptor Malignant neoplasm of lung Mediating Mediator Mus Mutate Non-Small-Cell Lung Carcinoma Oncogenic Outcome Parents Pathogenicity Population Predisposition Process Protocols documentation Respiratory Disease Retroelements Retrotransposition Retrotransposon Role Signal Transduction TP53 gene Technology Testing Toxic effect Validation Variant cancer type carcinogenicity combat constitutive expression design exposed human population functional genomics gene environment interaction genetic manipulation genome-wide in silico in vivo inducible gene expression insight male member mouse model novel therapeutics promoter renal epithelium respiratory health response single-cell RNA sequencing transcriptomics tumor

项目摘要

Abstract Functional genomics studies have unraveled many of the instructions encoded in the human genome. Taking advantage of significant advances in parallel sequencing and a wide diversity of other protocols, studies are proposed in this application to resolve gene-environment interactions that determine variations in the susceptibility to lung cancer. These studies are timely in light of the worsening levels of environmental pollution in many US cities that contribute to the development of respiratory disease and to disparities across populations. Of concern is the persistence of polycyclic aromatic hydrocarbon (PAH) contaminants, such as benzo(a)pyrene (BaP), and their ability to damage the DNA of lung epithelial cells. While the mutagenicity of BaP has been extensively characterized, questions remain about the degree to which DNA damage intersects with epigenetic disruption in driving the overwhelming carcinogenic response of BaP. Genome-wide assessments of BaP toxicity in the Ramos Laboratory revealed that the parent hydrocarbon and its metabolites activate LINE-1 retrotransposons in lung epithelial cells via epigenetic mechanisms. LINE-1 (Long Interspersed Element-1) activation is associated with oncogenic signaling, changes in chromatin structure, disruption of epigenetic control, and malignant transformation. LINE-1 is a family of mobile elements with the ability to copy their own DNA and use these complementary sequences to randomly insert at other locations within the genome. This process can be highly mutagenic and associated with genomic instability. We posit that alterations in the lifecycle of LINE-1 retroelements is a key missing link in our understanding of the complex genetic and epigenetic deficits associated with BaP carcinogenicity. Of ~100 full-length LINE-1 elements that remain competent for retrotransposition, nine are recognized as “hot” LINE-1s responsible for the vast majority of pathogenic events. These hot elements are polymorphic in humans and may therefore contribute to heterogeneity in genetic susceptibility to environmental PAH toxicity. In response to RFA-ES-20-018, we propose to use in vitro functional genomics for discovery and validation to test the hypothesis that polymorphic LINE-1 elements differentially regulate oncogenic signaling in lung epithelial cells and account for differences in susceptibility to BaP. In Aim 1, we will examine hot LINE-1 elements and their constitutive and inducible expression in normal lung and cancerous epithelial cells by targeted DNA sequencing and single cell RNA sequencing (scRNA-seq). In Aim 2, we will use CRISPR/dCas9m to edit the LINE-1 promoter to either enhance or silence retrotransposition and its impact on chromatin structure and transcriptomic landscapes. In Aim 3, we will use computational approaches to study genetic relationships across variable networks defined by polymorphic LINE-1 variants, with a focus on TP53, AHR, and RB. These studies will reveal important mechanistic insights into the activation of LINE-1 by PAH carcinogens and illuminate our understanding of the human variability in carcinogen susceptibility. This knowledge will lead to better biomarker designs and novel therapies to combat environmental toxicities.

抽象的功能基因组学研究揭示了人类基因组中编码的许多指令。利用并行测序方面的显着进步和其他各种方案的优势，研究正在本申请中提出解决基因与环境相互作用的问题，这些相互作用决定了鉴于环境污染程度日益恶化，这些研究是及时的。在美国许多城市，这导致了呼吸道疾病的发展和人口之间的差异。令人担忧的是多环芳烃 (PAH) 污染物的持续存在，例如苯并(a)芘 (BaP) 及其损伤肺上皮细胞 DNA 的能力，而 BaP 的致突变性已被证实。总体而言，DNA 损伤与表观遗传的交叉程度仍然存在疑问破坏 BaP 的全基因组毒性评估的压倒性致癌反应。拉莫斯实验室揭示母体碳氢化合物及其代谢物激活 LINE-1 通过表观遗传机制，肺上皮细胞中的逆转录转座子（长散布元件-1）。激活与致癌信号、染色质结构的变化、表观遗传学的破坏有关 LINE-1是一个具有复制自身能力的移动元素家族。 DNA 并使用这些互补序列随机插入基因组内的其他位置。我们认为生命周期的改变可能具有高度诱变性并与基因组不稳定性相关。 LINE-1逆转录因子的存在是我们理解复杂的遗传和表观遗传缺陷的关键缺失环节约 100 个仍具有能力的全长 LINE-1 元件。在逆转录转座中，有九个被认为是“热”LINE-1，负责绝大多数致病事件。这些热元素在人类中具有多态性，因此可能导致遗传异质性对环境 PAH 毒性的敏感性根据 RFA-ES-20-018，我们建议使用体外功能性。用于发现和验证的基因组学，以测试多态性 LINE-1 元件差异的假设调节肺上皮细胞中的致癌信号并解释对 BaP 的敏感性差异。 1，我们将检查热LINE-1元件及其在正常肺和肺中的组成型和诱导型表达。通过靶向 DNA 测序和单细胞 RNA 测序 (scRNA-seq) 检测癌性上皮细胞。我们将使用 CRISPR/dCas9m 编辑 LINE-1 启动子以增强或沉默逆转录转座及其对染色质结构和转录组景观的影响在目标 3 中，我们将使用计算方法。研究由多态 LINE-1 变体定义的可变网络之间的遗传关系，重点是 TP53、AHR 和 RB 这些研究将揭示 LINE-1 激活的重要机制。 PAH 致癌物并阐明了我们对人类致癌物易感性差异的理解。知识将带来更好的生物标志物设计和对抗环境毒性的新疗法。