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 转座元素是人类遗传变异的主要来源，也通过引起体细胞突变来促进人类疾病。人类癌症中转置的活性已建立。由L1元素编码的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普遍。然而，逆转录置位是否以及如何促进癌症基因组进化并促进肿瘤发生尚不清楚。在这里，我们假设逆转录置位可能通过插入外引起远程基因组重排而促进癌症的进化。该假设是基于原发性癌症基因组中的基因组观察以及我们体外实验的分子和基因组数据的提出的。为了检验这一假设，我们将在体外模型中（AIM 1）中的实验方法与逆转录置位和反转座相关的重排的计算分析（AIM 2）（AIM 2）（AIM 2）（AIM 2）（AIM 2）。具体而言，我们提出，线1插入的非传统分辨率会导致DNA断裂和远程DNA重排，并且这些重排的子集通过产生厌食或二分化染色体，从而进一步破坏了基因组，从而经历重新排列的波浪。为了测试这一点，我们首先评估DNA损伤及其对L1 ORF2P（AIM 1A）的核酸内切酶（EN）和逆转录酶（RT）活性的依赖性。然后，我们使用将细胞生物学和全基因组测序结合的最先进的look-seq方法（AIM 1B和1C）确定了ORF2P诱导的DNA损伤的直接和下游基因组后果。通过补充逆转录置术的这些体外分析，我们建议分析大量的癌症基因组（AIM 2B）和单个癌细胞细胞基因组（AIM 2C），以确定评估插入和远距离重排的普遍性，这些重排具有转化转化的标志。我们专注于逆转录术患病率极高的食管癌（在> 90％的癌症中），而被称为Barrett的食管（BE）的预癌状况也很高，此前还观察到Line-1逆转录置位。拟议的工作包括开发用于shot弹枪癌测序数据中的联合重排和转位检测的新生物信息学包装，这将使研究遗传变异，基因组不稳定性和体细胞基因组进化的研究人员有益于研究人员（AIM 2A）。共同的研究将彻底表征line-1逆转录位置与基因组不稳定性之间的关系，以及逆转录位置在癌症进化中的作用。这些知识可能导致预防癌症和治疗剂的新途径。