Consequences of retrotransposition on genome integrity

逆转录转座对基因组完整性的影响

• 批准号: 10736406
• 负责人: KATHLEEN H BURNS
• 金额: $ 77.07万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736406
• 关键词: Barrett Esophagus; Bioinformatics; Biological; Burn injury; Cells; Cellular biology; Chromosomal Instability; Chromosomes; Clonal Expansion; Colon Carcinoma; Complement; Complementary DNA; Computer Analysis; DNA; DNA Damage; DNA Repair; DNA Sequence Rearrangement; DNA Transposable Elements; DNA copy number; DNA sequencing; Data; Data Set; Dependence; Detection; Development; Dicentric chromosome; Elements; Esophageal Adenocarcinoma; Event; Evolution; Experimental Models; Gene Rearrangement; Genes; Genetic Screening; Genetic Transcription; Genetic Variation; Genetic study; Genome; Genomic Instability; Genomics; High Prevalence; Human; Human Genetics; In Vitro; Insertion Mutation; Joints; Knowledge; Laboratories; Lesion; Long Interspersed Elements; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Mediating; Methods; Methylation; Modeling; Molecular; Molecular Biology; Monitor; Mutation; Open Reading Frames; Outcome; Pathology; Population; Prevalence; Process; Proteins; RNA; RNA-Binding Proteins; RNA-Directed DNA Polymerase; Research Personnel; Resolution; Retrotransposition; Retrotransposon; Reverse Transcription; Role; S phase; Shotguns; Somatic Mutation; Source; System; Testing; The Cancer Genome Atlas; Therapeutic; Tissues; Work; analytical method; cancer cell; cancer genome; cancer prevention; cancer type; endonuclease; experimental study; genome analysis; genome integrity; genome sequencing; genomic data; human disease; human tissue; in vitro Model; insight; live cell imaging; malignant stomach neoplasm; metaplastic cell transformation; novel strategies; overexpression; premalignant; promoter; replication stress; tumor; tumorigenesis; whole genome

Project Summary Transposable elements are a major source of human genetic variation and also contribute to human diseases by causing somatic mutations. The activity of transposition in human cancers is well established. Both expression of the RNA binding protein ORF1p encoded by the L1 element and insertions of L1 and other transposons are commonplace in cancer cells and cancer genomes. However, whether and how retrotransposition contributes to cancer genome evolution and promotes tumorigenesis remain unclear. Here, we hypothesize that retrotransposition may promote cancer evolution by causing long-range genomic rearrangements in addition to insertions. This hypothesis is formulated based on both genomic observations in primary cancer genomes and molecular and genomic data from our in vitro experiments. To test this hypothesis, we will combine experimental approaches in in vitro models (Aim 1) with computational analyses (Aim 2) of retrotransposition and retrotransposition-associated rearrangements in primary cancers and precancer cells (Aim 2). Specifically, we propose that non-canonical resolution of LINE-1 insertions can cause DNA breaks and long-range DNA rearrangements, and a subset of these rearrangements further destabilizes genomes by generating acentric or dicentric chromosomes that undergo waves of rearrangements. To test this, we first assess DNA damage and its dependence on the endonuclease (EN) and reverse transcriptase (RT) activities of L1 ORF2p (Aim 1a). We then determine both immediate and downstream genomic consequences of ORF2p-induced DNA damage using state-of-the-art Look-Seq approaches that combine cell biology and whole-genome sequencing (Aim 1b and 1c). Complementing these in vitro analyses of retrotransposition, we propose to analyze bulk cancer genomes (Aim 2b) and single pre-cancer cell genomes (Aim 2c) to definitively assess the prevalence of insertions and long-range rearrangements that bear the hallmarks of retrotransposition. We focus on esophageal cancers with an extremely high prevalence of retrotransposition (in >90% of cancers) and the precancer condition known as Barrett's Esophagus (BE), where LINE-1 retrotransposition has also been observed previously. The proposed work includes development of new bioinformatic packages for joint rearrangement and transposition detection in shotgun cancer sequencing data that will benefit researchers studying genetic variation, genome instability, and somatic genome evolution (Aim 2a). Together, the proposed studies will thoroughly characterize the relationships between LINE-1 retrotransposition and genome instability writ large and the roles of retrotransposition in cancer evolution. This knowledge may lead to new avenues of cancer prevention and therapeutics.

项目概要 转座因子是人类遗传变异的主要来源，也通过引起体细胞突变而导致人类疾病。转座在人类癌症中的活性已得到充分证实。 L1 元件编码的 RNA 结合蛋白 ORF1p 的表达以及 L1 和其他转座子的插入在癌细胞和癌症基因组中都很常见。然而，逆转录转座是否以及如何促进癌症基因组进化并促进肿瘤发生仍不清楚。在这里，我们假设逆转录转座除了插入之外还可能通过引起长程基因组重排来促进癌症进化。这一假设是基于对原发性癌症基因组的基因组观察以及来自我们体外实验的分子和基因组数据而制定的。为了检验这一假设，我们将结合体外模型的实验方法（目标 1）与原发性癌症和癌前细胞中逆转录转座和逆转录转座相关重排的计算分析（目标 2）（目标 2）。具体来说，我们认为 LINE-1 插入的非规范分辨率可能会导致 DNA 断裂和长程 DNA 重排，并且这些重排的一部分通过产生经历重排波的无着丝粒或双着丝粒染色体来进一步破坏基因组的稳定性。为了测试这一点，我们首先评估 DNA 损伤及其对 L1 ORF2p 的核酸内切酶 (EN) 和逆转录酶 (RT) 活性的依赖性（目标 1a）。然后，我们使用结合细胞生物学和全基因组测序的最先进的 Look-Seq 方法确定 ORF2p 诱导的 DNA 损伤的直接和下游基因组后果（目标 1b 和 1c）。作为逆转录转座体外分析的补充，我们建议分析大量癌症基因组 (Aim 2b) 和单个癌前细胞基因组 (Aim 2c)，以明确评估具有逆转录转座标志的插入和长程重排的发生率。我们重点关注逆转录转座发生率极高的食管癌（>90% 的癌症）和被称为巴雷特食管 (BE) 的癌前病变，此前也观察到过 LINE-1 逆转录转位。拟议的工作包括开发新的生物信息学软件包，用于鸟枪法癌症测序数据中的联合重排和转座检测，这将有利于研究遗传变异、基因组不稳定性和体细胞基因组进化的研究人员（目标 2a）。总之，拟议的研究将彻底描述 LINE-1 逆转录转座和基因组不稳定性之间的关系以及逆转录转座在癌症进化中的作用。这些知识可能会带来癌症预防和治疗的新途径。