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.

摘要 功能基因组学研究已经揭示了人类基因组中编码的许多指令。以 利用平行测序的重大进展和其他协议的广泛多样性，研究 在本申请中提出的解决基因-环境相互作用，决定在基因的变化， 对肺癌的易感性。鉴于环境污染日益严重，这些研究是适时的 在许多美国城市，这有助于呼吸系统疾病的发展和人口之间的差距。 令人关切的是多环芳烃污染物的持久性，如苯并（a）芘 (BaP)以及它们破坏肺上皮细胞DNA的能力。虽然苯并（a）芘的致突变性已被 尽管DNA损伤的特征得到了广泛的表征，但关于DNA损伤与表观遗传学的交叉程度仍存在疑问。 破坏了苯并（a）芘的压倒性致癌反应。BaP毒性的全基因组评估 在拉莫斯实验室的研究表明，母体碳氢化合物及其代谢物激活LINE-1 逆转录转座子通过表观遗传机制在肺上皮细胞中的表达。LINE-1（长间隔元素-1） 激活与致癌信号传导、染色质结构的变化、表观遗传学的破坏、 控制和恶性转化。LINE-1是一系列移动的元素，具有复制自己元素的能力 DNA并使用这些互补序列随机插入基因组内的其他位置。这 这一过程可能是高度致突变的，并与基因组不稳定性有关。我们认为生命周期的改变 LINE-1逆转录因子是我们理解复杂遗传和表观遗传缺陷的关键缺失环节 与BaP致癌性有关。在大约100个全长LINE-1元件中， 在逆转录转座中，九个被认为是负责绝大多数致病事件的“热”LINE-1。 这些热元件在人类中是多态的，因此可能有助于遗传异质性。 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 基因组学的发现和验证，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敏感性的差异。在Aim中 1，我们将检查热LINE-1元件及其在正常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）检测癌上皮细胞。在目标2中， 我们将使用CRISPR/dCas 9 m来编辑LINE-1启动子，以增强或沉默逆转录转座及其表达。 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 研究由多态性LINE-1变体定义的可变网络之间的遗传关系，重点是 TP 53、AHR和RB。这些研究将揭示LINE-1激活的重要机制， 多环芳烃致癌物和照亮我们的理解，在致癌物的易感性的人类变异。这 知识将导致更好的生物标志物设计和新的治疗方法，以对抗环境毒性。