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

G-四链体介导的 PDGFR-?? 转录调控

• 批准号: 8657680
• 负责人: LAURENCE H. HURLEY
• 金额: $ 5.08万
• 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/8657680
• 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; 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; screening; 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.

描述（由申请人提供）：PDGFR-β的过表达是胰腺癌以及其他炎性疾病（包括动脉硬化）中的重要因素，因此是非常重要的分子靶标。该提案的长期目标是开发特异性抑制PDGFR-β基因表达的小分子治疗剂。我们的策略利用了最近对G-四链体在包括c-Myc在内的许多基因的转录沉默中的重要性的认识。本提案中报告的初步数据表明，一组四个重叠的G-四链体是控制PDGFR-β转录的关键元件。我们还证明了已知的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，这取决于对组成G-四链体的选择性。转录诱导的超螺旋性在启动子区双链体DNA向G-四链体的转化过程中起着重要作用。因此，我们要测试的假设是，转录诱导的负超螺旋性提供了一个实时反馈机制，进入PDGFR-β启动子中的NHE，以调节PDGFR-β转录的激发速率（巡航控制）和激活或沉默（开/关开关）。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、34中和34端G-四链体形成序列的生物学功能，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启动子中的组成性G-四链体差异结合的特异性蛋白质和小分子。对于具体目标1，我们将构建含有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，高场NMR将用于确定位于PDGFR-β促进剂元件中的组成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。