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