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; 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揭示了人类基因组中编码的许多指令。服用 平行测序和其他各种方案的多样性的显着进步的优势，研究是 在本应用中提出的以解决确定基因环境的相互作用，以确定 对肺癌的敏感性。考虑到令人担忧的环境污染水平，这些研究是及时的 在许多美国城市，有助于呼吸道疾病的发展和跨种群的差异。 令人担忧的是多环芳烃（PAH）污染物的持久性，例如苯并（a）pyrene （BAP）及其损害肺上皮细胞DNA的能力。 bap的诱变性已经 广泛的表征，关于DNA损伤与表观遗传相交的程度仍然存在问题 驱动BAP压倒性的致癌反应的破坏。 BAP毒性的全基因组评估 在拉莫斯实验室中，父母碳氢化合物及其代谢产物激活线路1 通过表观遗传机制在肺上皮细胞中的逆转录子。 LINE-1（长长的元素1） 激活与致癌信号传导，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是一个手机元素的家庭，能够复制自己的家族 DNA并使用这些完成序列在基因组中的其他位置随机插入。这 过程可能是高度诱变的，并且与基因组不稳定性有关。我们指出生命周期的改变 LINE-1的恢复元素是我们对复杂遗传和表观遗传缺陷的理解中的关键缺失链接 与BAP致癌性有关。在〜100个全长线1元素中仍然有能力 逆转录位，九个被认为是导致绝大多数致病事件的“热”线路1。 这些热元素在人类中是多态性的，因此可能导致遗传的异质性 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，以检验多态线1元素差异化的假设 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。目标 1，我们将检查热线1元素及其在正常肺和 通过靶向DNA测序和单细胞RNA测序（SCRNA-SEQ）癌上皮细胞。在AIM 2中， 我们将使用CRISPR/DCAS9M编辑Line-1启动子，以增强或沉默返回转录及其 在AIM 3中，我们将使用计算方法 研究由多态线路1变体定义的可变网络的遗传关系，重点是 TP53，AHR和RB。这些研究将揭示对线路1激活的重要机理见解 PAH致癌物并阐明了我们对致癌敏感性人类变异性的理解。这 知识将导致更好的生物标志物设计和新颖的疗法来打击环境毒性。