G-Quadruplex-Mediated Transcriptional Regulation of PDGFR-??

G-四链体介导的 PDGFR-?? 转录调控

• 批准号: 8287242
• 负责人: LAURENCE H. HURLEY
• 金额: $ 5.41万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8287242
• 关键词: Adenine; Affinity Chromatography; Arteriosclerosis; Aspergillus Nuclease S1; Base Pairing; Beryllium; Binding; Biological; Biological Assay; Biological Process; DNA; Data Reporting; Dependency; Disease; Dose; Elements; Feedback; Functional disorder; G-Quartets; GC Rich Sequence; Gene Expression; Genes; Genetic Transcription; Goals; Guanine; Housing; Human; Indium; Individual; Inflammatory; Libraries; Luciferases; Malignant Neoplasms; Malignant neoplasm of pancreas; Maps; Mediating; Methods; Molecular Target; Mus; Mutation; Nuclear Extract; PDGFRB gene; Pattern; Pharmaceutical Preparations; Plasmids; Platelet-Derived Growth Factor Receptor; Platelet-Derived Growth Factor beta Receptor; Primer Extension; Promoter Regions; Proteins; Relative (related person); Reporter; Reporter Genes; Running; Screening procedure; Sequence Deletion; Sp1 Transcription Factor; Structure; System; Techniques; Testing; Therapeutic; Time; Transcription Initiation Site; Transcriptional Regulation; base; c-myc Genes; combinatorial; insight; mutant; nuclease; overexpression; programs; promoter; public health relevance; research study; small molecule; structural biology; telomestatin; two-dimensional

DESCRIPTION (provided by applicant): Overexpression of PDGFR-beta is an important factor in pancreatic cancer as well as other inflammatory diseases, including arteriosclerosis and therefore is a highly significant molecular target. The long-range goal of this proposal is to develop small molecule therapeutics that will specifically suppress PDGFR-beta gene expression. Our strategy takes advantage of the recent insight into the importance of G-quadruplexes in transcriptional silencing of a number of genes, including c-Myc. Preliminary data reported in this proposal demonstrate that a cluster of four overlapping G-quadruplexes are key elements in the control of PDGFR-beta transcription. We have also demonstrated that known G-quadruplex-interactive compounds have differential effects on PDGFR-beta gene expression dependent on the selectivity for the constituent G-quadruplexes. Transcriptionally induced superhelicity has been demonstrated to be important in the conversion of duplex DNA to G-quadruplex in promoter region. Thus our hypothesis to be tested is that the negative superhelicity induced by transcription provides a real-time feedback mechanism into the NHE in the PDGFR-beta promoter to modulate both the firing rate (cruise control) and activation or silencing (on/off switch) of PDGFR-beta transcription. The specific aims are: (1) To determine the biological function of the 54-end, mid-54, mid-34, and 34-end G-quadruplex-forming sequences by deletion and mutational analysis of the PDGFR-beta promoter element and then determine the effect of supercoiling on the pattern of G-quadruplex formation in the PDGFR-beta promoter. (2) To determine by NMR the folding patterns and structures of the G-quadruplexes in the PDGFR-beta promoter. (3) To identify specific proteins and small molecules that bind differentially to the constituent G-quadruplexes found in the PDGFR-beta promoter. For specific aim 1, we will construct supercoiled plasmids containing PDGFR-beta promoter inserts and a luciferase reporter system. For specific aim 2, high-field NMR will be used to determine the structures of the constituent G-quadruplexes located in the PDGFR-beta promoter element. For specific aim 3, we will use affinity chromatography and small molecule screening methods to identify proteins and drug-like molecules that bind to the individual G-quadruplexes. The biological effects of these entities will be determined in specific aim 1. PUBLIC HEALTH RELEVANCE: An effective means to turn on and off genes associated with diseases, such as cancer and arteriosclerosis, with small drug-like molecules is still not available. In this project, we take advantage of a new mechanism to achieve this objective. The target is a gene that is commonly involved in pancreatic cancer and arteriosclerosis.

描述（由申请人提供）：PDGFR-β的过度表达是胰腺癌以及其他炎症性疾病（包括动脉硬化）的重要因素，因此是非常重要的分子靶点。该提案的长期目标是开发特异性抑制 PDGFR-β 基因表达的小分子疗法。我们的策略利用了最近对 G-四链体在包括 c-Myc 在内的许多基因转录沉默中的重要性的见解。该提案中报告的初步数据表明，四个重叠的 G-四链体簇是控制 PDGFR-β 转录的关键元件。我们还证明，已知的 G-四链体相互作用化合物对 PDGFR-β 基因表达具有不同的影响，具体取决于组成 G-四链体的选择性。转录诱导的超螺旋性已被证明对于启动子区域双链体 DNA 向 G-四链体的转化非常重要。因此，我们要测试的假设是，转录诱导的负超螺旋向 PDGFR-β 启动子中的 NHE 提供实时反馈机制，以调节发射速率（巡航控制）和激活或沉默（开/关开关） PDGFR-β 转录。具体目标是：（1）通过PDGFR-β启动子元件的缺失和突变分析，确定54端、54中端、34中端和34端G四链体形成序列的生物学功能，以及然后确定超螺旋对 PDGFR-β 启动子中 G-四链体形成模式的影响。 (2)通过NMR确定PDGFR-β启动子中G-四链体的折叠模式和结构。 (3) 鉴定与 PDGFR-β 启动子中发现的 G-四链体成分有差异结合的特定蛋白质和小分子。对于具体目标 1，我们将构建包含 PDGFR-β 启动子插入片段和荧光素酶报告系统的超螺旋质粒。对于具体目标 2，将使用高场 NMR 来确定位于 PDGFR-β 启动子元件中的组成 G-四链体的结构。对于具体目标 3，我们将使用亲和层析和小分子筛选方法来鉴定与单个 G-四链体结合的蛋白质和药物样分子。这些实体的生物效应将在具体目标 1 中确定。 公共健康相关性：目前还没有一种有效的方法可以用类似药物的小分子来打开和关闭与癌症和动脉硬化等疾病相关的基因。在这个项目中，我们利用一种新机制来实现这一目标。目标是通常与胰腺癌和动脉硬化有关的基因。