Functional Genomics of LINE-1 Retrotransposition

LINE-1 逆转录转座的功能基因组学

• 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
• 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

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 功能基因组学研究已经解开了人类基因组中编码的许多指令。服用 利用并行测序的重大进展和各种其他方案的优势，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 患肺癌的易感性。鉴于环境污染日益严重，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。虽然 BaP 的致突变性已被 尽管已得到广泛表征，但关于 DNA 损伤与表观遗传的交叉程度仍然存在疑问 破坏 BaP 的压倒性致癌反应。 BaP 毒性的全基因组评估 拉莫斯实验室揭示母体碳氢化合物及其代谢物激活 LINE-1 肺上皮细胞中的逆转录转座子通过表观遗传机制。 LINE-1（长散布元素-1） 激活与致癌信号、染色质结构的变化、表观遗传的破坏有关 控制和恶性转化。 LINE-1 是一系列移动元素，能够复制自己的元素 DNA 并使用这些互补序列随机插入基因组内的其他位置。这 该过程可能具有高度诱变性并与基因组不稳定相关。我们假设生命周期的改变 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 与 BaP 致癌性有关。约 100 个仍能胜任的全长 LINE-1 元素 在逆转录转座中，有 9 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。为了响应 RFA-ES-20-018，我们建议使用体外功能 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传导并解释对 BaP 敏感性的差异。瞄准 1，我们将检查热LINE-1元件及其在正常肺和组织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。在目标 2 中， 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。这些研究将通过以下方式揭示 LINE-1 激活的重要机制见解： PAH 致癌物并阐明了我们对人类致癌物易感性差异的理解。这 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。