Testing the Polyomavirus-based Replication Dependent Enhancer Duplication Model

测试基于多瘤病毒的复制依赖性增强子复制模型

• 批准号: 10510138
• 负责人: PETER Augustus BULLOCK
• 金额: $ 20.63万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-14 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10510138
• 关键词: Address; Biological Assay; Biological Models; Cell Line; Cells; Cellular Assay; DNA; DNA biosynthesis; Detection; Disease; Double Strand Break Repair; Doxycycline; Enhancers; Enzymatic Biochemistry; Event; Frequencies; GTP-Binding Protein alpha Subunits, Gs; Genes; Genome; Goals; Human; Human Cell Line; Immune; Immunocompromised Host; Individual; Malignant Neoplasms; Maturation-Promoting Factor; Modeling; Monitor; Nucleic Acid Regulatory Sequences; Nucleosomes; Okazaki fragments; Outcome; Pathogenesis; Pathogenicity; Patients; Plasmids; Play; Polyomavirus; Positioning Attribute; Primates; Proteins; RTH-1 Nuclease; Reaction; Role; Simian virus 40; Small Interfering RNA; Source; Surgical Flaps; System; Techniques; Testing; Transfection; Tumor Suppressor Genes; Update; Viral; Virus Replication; base; cell type; design; detection assay; experimental study; human disease; interest; knock-down; next generation sequencing; patient population; predictive modeling; small hairpin RNA; theories; viral DNA

When purified from healthy individuals, the regulatory regions of most human polyomaviruses contain single enhancers. However, when isolated from productively infected cells from immune- compromised individuals, the genomes of polyomaviruses contain duplicated enhancers that promote viral DNA replication and subsequent pathogenesis. The mechanism(s) that give rise to the duplicated enhancers in pathogenic polyomaviruses are, however, not known. To address this issue, we proposed a model for the duplication of the polyomavirus enhancers that is based upon advances in our understanding of; 1) the initiation of polyomavirus DNA replication, 2) the formation of “long flaps” via displacement synthesis o f O k a z a k i f r a g m e n t s and 3) the subsequent steps needed for double stranded break repair. We now wish to test key features of the “Replication Dependent Enhancer Duplication” (RDED) model. Regarding the assays that will be employed to test this model, previous experiments established that the enhancer duplications can be detected in CV1 cells following transfection of SV40 virus. Thus, this is a viable assay for testing the RDED model. However, we wish to test the hypothesis that the enhancer duplications will also occur in more versatile plasmid-based replication assays. Once the optimal cell type and assays (e.g., PCR) are established, we will use Next-generation sequencing (NGS) to quantitate the frequency of the enhancer duplications and to identify the sequences of all the products formed in the reactions. In addition, we will test the RDED- based hypothesis that the enhancer duplications will be promoted by the aberrant expression of factors needed for Okazaki fragment formation. For instance, a cell line containing a doxycycline inducible shRNA targeting FEN-1 will be used to test the hypothesis that the resulting “long flap” containing nascent DNA will promote the enhancer duplications. In related experiments, we will test the hypothesis that hairpins designed to block FEN1 loading will also promote the enhancer duplications. Additional experiments will test the RDED-based hypothesis that nicks are generated on the template strand in the vicinity of the viral origin. Moreover, we will test the theory that nucleosomes play a major role in limiting the enhancer duplications to the regulatory region. Finally, non-viral enhancers are also subjected to duplications and deletions and these alterations significantly impact the activity of disease genes, including oncogenes and tumor suppressors. Therefore, our studies of the formation of the enhancer duplications in polyomaviruses will have an important role in establishing the mechanism(s) needed to form the enhancer duplications associated with a wide variety of human diseases.

当从健康个体纯化时，大多数人多瘤病毒的调节区含有单个增强子。然而，当从来自免疫受损个体的生产性感染细胞中分离时，多瘤病毒的基因组含有促进病毒DNA复制和随后发病的重复增强子。然而，致病性多瘤病毒中产生复制增强子的机制尚不清楚。为了解决这个问题，我们提出了一种多瘤病毒增强子复制的模型，该模型基于我们对以下方面的理解的进展：1）多瘤病毒DNA复制的起始，2）通过Ok a z a k i f r a g m e n t s的置换合成形成“长瓣”，和3）双链断裂修复所需的后续步骤。我们现在希望测试“复制依赖增强子复制”（RDED）模型的关键特征。关于将用于测试该模型的测定，先前的实验确定了在SV 40病毒转染后可以在CV1细胞中检测到增强子复制。因此，这是一种用于检测RDED模型的可行试验。然而，我们希望测试的假设，增强子复制也将发生在更通用的质粒为基础的复制测定。一旦最佳细胞类型和测定（例如，PCR）建立后，我们将使用下一代测序（NGS）来定量增强子复制的频率，并鉴定反应中形成的所有产物的序列。此外，我们将测试基于RDED的假设，即增强子复制将由冈崎片段形成所需的因子的异常表达促进。例如，含有靶向FEN-1的多西环素诱导型shRNA的细胞系将用于测试所得到的含有新生DNA的“长瓣”将促进增强子复制的假设。在相关的实验中，我们将测试这样的假设，即设计用于阻断FEN 1加载的发夹也会促进增强子复制。其他实验将检验基于RDED的假设，即在病毒来源附近的模板链上产生切口。此外，我们将测试核小体在限制增强子复制到调控区中起主要作用的理论。最后，非病毒增强子也会发生复制和缺失，这些改变会显著影响疾病基因的活性，包括癌基因和肿瘤抑制基因。因此，我们对多瘤病毒中增强子重复形成的研究将在建立形成与多种人类疾病相关的增强子重复所需的机制方面发挥重要作用。