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.

项目摘要 转座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。在人类癌症中，转位的活性已经得到了很好的证实。由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是常见的。然而，逆转录转座是否以及如何有助于癌症基因组的进化和促进肿瘤的发生仍不清楚。在这里，我们假设逆转座可能通过引起插入以外的远距离基因组重排来促进癌症的进化。这一假说是基于对原发癌症基因组的基因组观察以及我们体外实验的分子和基因组数据而形成的。为了验证这一假设，我们将结合体外模型中的实验方法(目标1)和计算分析(目标2)原发癌和癌前细胞中的逆转位和逆转位相关重排(目标2)。具体地说，我们认为LINE-1插入的非规范分辨可以导致DNA断裂和远程DNA重排，这些重排的子集通过产生经历重排浪潮的无着丝粒或双着丝粒染色体而进一步破坏基因组的稳定。为了验证这一点，我们首先评估了DNA损伤及其对L1 ORF2p(目标1a)的内切酶(EN)和逆转录酶(RT)活性的依赖。然后，我们使用结合了细胞生物学和全基因组测序的最先进的Look-Seq方法来确定ORF2p诱导的DNA损伤的直接和下游基因组后果(目标1b和1c)。为了补充这些逆转录转座的体外分析，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)，以明确评估带有逆转录转座特征的插入和远程重排的流行率。我们关注逆转位发生率极高的食道癌(占癌症的90%)和被称为Barrett‘s食道(BE)的癌前疾病，在此之前也观察到过LINE-1逆转位。拟议的工作包括开发新的生物信息学包，用于在鸟枪式癌症测序数据中进行联合重排和转位检测，这将有助于研究遗传变异、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，拟议的研究将彻底表征LINE-1逆转座和基因组不稳定性之间的关系，以及逆转座在癌症进化中的作用。这一知识可能会导致癌症预防和治疗的新途径。