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.

当从健康个体中纯化时，大多数人类多瘤病毒的调节区域都包含单个增强剂。但是，当从免疫受损的个体中从有效感染的细胞中分离出来时，多瘤病毒的基因组含有重复的增强子，可促进病毒DNA复制和随后的发病机理。然而，尚不清楚导致致病性多瘤病毒中重复增强子的机制。为了解决这个问题，我们提出了一个基于我们对理解的进步的多瘤病毒增强剂的复制模型； 1）多瘤病毒DNA复制的主动性，2）通过位移合成的“长襟翼”形成了“长皮瓣”，与双绞合断裂修复所需的后续步骤。我们现在希望测试“依赖于复制的增强器重复”（RDED）模型的关键功能。关于将员工测试该模型的攻击，先前的实验表明，在SV40病毒转化后，可以在CV1细胞中检测到增强子的重复。这是测试RDED模型的可行测定法。但是，我们希望检验以下假设：增强子的重复也将在更通用的基于质粒的复制测定中发生。一旦建立了最佳细胞类型和测定（例如PCR），我们将使用下一代测序（NGS）来量化增强子重复的频率并确定反应中所有产物的序列。此外，我们还将检验基于RD的假设，即增强子的重复将通过Okazaki碎片形成所需的因素的异常表达来促进。例如，含有强力霉素诱导的shRNA靶向FEN-1的细胞系将用于检验以下假设：所得的“长瓣”含有新生的DNA将促进增强子的重复。在相关的实验中，我们将测试以下假设：旨在阻止Fen1载荷的发夹也将促进增强子的重复。其他实验将测试基于RD的假设，即在病毒起源附近的模板链上产生了划痕。此外，我们将测试核小体在将增强子重复限制到调节区域中起主要作用的理论。最后，非病毒增强剂还受到重复和缺失，这些改变显着影响疾病基因的活性，包括癌基因和肿瘤补充剂。因此，我们对多瘤病毒中增强子重复的形成的研究将在建立形成与各种各样人类疾病相关的增强子重复所需的机制中起重要作用。