Functional Genomics of LINE-1 Retrotransposition

LINE-1 逆转录转座的功能基因组学

• 批准号: 10337555
• 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/10337555
• 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 Adducts; DNA Damage; DNA Methylation; DNA sequencing; Development; Disease; Elements; 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; Light; Link; Liver; Location; Long Interspersed Elements; Lung; Malignant - descriptor; Malignant neoplasm of lung; Mediating; Mediator of activation protein; 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; Time; Toxic effect; Validation; Variant; base; cancer type; carcinogenicity; combat; design; exposed human population; functional genomics; gene environment interaction; genetic manipulation; genome-wide; in silico; in vivo; inducible gene expression; insertion/deletion mutation; 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.

摘要 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。拿走 利用并行测序的重大进展和广泛多样的其他方案，研究包括 在本应用程序中提出，以解决基因-环境交互作用，这些交互作用决定了 易患肺癌。鉴于环境污染的恶化程度，这些研究是适时的。 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。 令人关切的是多环芳烃(PAH)污染物的持久性，如苯并(A)芘 (BaP)，以及它们损伤肺上皮细胞DNA的能力。虽然BaP的致突变性已经被 被广泛描述的问题仍然是DNA损伤与表观遗传的交叉程度 破坏了BaP的压倒性致癌反应。全基因组对苯并苯毒性的评估 拉莫斯实验室发现，母体碳氢化合物及其代谢产物激活了LINE-1 肺上皮细胞中的反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、表观遗传学的中断有关 控制和恶变。Line-1是一系列移动元素，能够复制自己的元素 并使用这些互补序列在基因组中的其他位置随机插入。这 这一过程可能是高度诱变的，并与基因组的不稳定性有关。我们假设生命周期中的变化 LINE-1逆转录元件是我们理解复杂的遗传和表观遗传缺陷的关键缺失的一环 与BaP的致癌性有关。-1\f25~100个全长-1\f25 LINE-1\f6元件 逆转录转座，其中9条被认为是导致绝大多数致病事件的“热线”。 这些热点元素在人类中是多态的，因此可能导致基因的异质性 对环境多环芳烃毒性的敏感性。为了响应RFA-ES-20-018，我们建议使用体外功能 发现和验证基因组学以检验多态LINE-1元件存在差异的假设 调节肺上皮细胞的致癌信号，并解释对BaP易感性的差异。在AIM 1，我们将检测热线-1元件及其在正常肺和正常肺中的结构性和诱导性表达 靶向DNA测序和单细胞RNA测序(scRNA-seq)。在目标2中， 我们将使用CRISPR/dCas9m编辑LINE-1启动子，以增强或抑制逆转录转座及其 对染色质结构和转录景观的影响。在目标3中，我们将使用计算方法 研究由多态LINE-1变异体定义的不同网络间的遗传关系，重点是 TP53、AHR和RB。这些研究将揭示对LINE-1激活的重要机制见解 多环芳烃是致癌物，并阐明了我们对人类致癌物易感性的变异性的理解。这 知识将导致更好的生物标记物设计和抗击环境毒性的新疗法。