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

G-四链体介导的 PDGFR-?? 转录调控

• 批准号: 8257544
• 负责人: LAURENCE H. HURLEY
• 金额: $ 35.11万
• 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/8257544
• 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(申请人提供)：PDGFR-β的过度表达是胰腺癌和包括动脉硬化在内的其他炎症性疾病的一个重要因素，因此是一个非常重要的分子靶点。这项提议的长期目标是开发能够特异性抑制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洞察。这项研究报告的初步数据表明，四个重叠的G-四联体是控制PDGFR-β转录的关键元件。我们还证明了已知的G-四链相互作用化合物对PDGFR-β基因的表达有不同的影响，这取决于组成G-四链的选择性。转录诱导的超螺旋作用在启动子区域双链DNA向G-四链的转化过程中起着重要作用。因此，我们要检验的假设是，转录诱导的负超螺旋为PDGFR-β启动子中的NHE提供了一种实时反馈机制，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)。其具体目的是：(1)通过对PDGFR-β启动子元件的缺失和突变分析，确定54-端、54-中期、34-中期和34-端G-四链形成序列的生物学功能，进而确定超螺旋对PDGFR-β启动子中G-四链形成模式的影响。(2)用核磁共振方法确定PDGFR-β启动子中G-四链的折叠模式和结构。(3)鉴定与PDGFR-β启动子中的G-四链体不同结合的特定蛋白质和小分子。针对特定的目的1，我们将构建含有PDGFR-β启动子插入和荧光素酶报告系统的超螺旋载体。对于特定的目的2，将使用高场核磁共振来确定位于PDGFR-β启动子元件中的组成G-四链的结构。对于特定的目标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-四链结合的蛋白质和类药物分子。这些实体的生物效应将在具体目标1中确定。 与公共卫生相关：目前还没有一种有效的方法来打开和关闭与疾病相关的基因，如癌症和动脉硬化，使用类似药物的小分子。在这个项目中，我们利用一种新的机制来实现这一目标。靶点是一种通常与胰腺癌和动脉硬化有关的基因。