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

G-四链体介导的 PDGFR-?? 转录调控

基本信息

批准号：
8450187
负责人：
LAURENCE H. HURLEY
金额：
$ 32.85万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
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/8450187
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

项目摘要

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症性疾病（包括动脉硬化）的重要因素，因此是一个非常重要的分子靶标。该提案的远程目标是开发小分子疗法，这些疗法将特别抑制PDGFR-beta基因表达。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力。该提案中报告的初步数据表明，四个重叠的G四链体的集群是控制PDGFR-Beta转录的关键要素。我们还证明，已知的G四链体相互作用化合物对pDGFR-beta基因表达具有不同的影响，取决于成分G-四链体的选择性。已证明转录诱导的超螺旋性在启动子区域的双链DNA转化为G-四链体中很重要。因此，我们要检验的假设是，转录引起的负超固有性为PDGFR-Beta启动子中的NHE提供了实时反馈机制，以调节PDGFR-BETA转录的发射速率（巡航控制）和激活或沉默（ON/OFF开关）。具体目的是：（1）通过缺失和PDGFR-BETA启动子元素的缺失和突变分析来确定54端，54，34中期和34端G Quadruplex形成序列的生物学功能，然后确定超涂层对G- Ququlexruplex构造在PDGGRERIN the PDGGREFREFRERERERERERIN in pDGGERERINIC inta PDGGRETA启动的效果。（2）通过NMR确定pDGFR-beta启动子中G-四链体的折叠模式和结构。（3）鉴定特定的蛋白质和小分子，它们与PDGFR-beta启动子中发现的成分G-四链体的结合。对于特定的目标1，我们将构建包含PDGFR-BETA启动子插入物和荧光素酶报告器系统的超螺旋质粒。对于特定目标2，将使用高场NMR来确定位于PDGFR-Beta启动子元件中的组成型G-四链体的结构。对于特定的目标3，我们将使用亲和色谱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。这些实体的生物学效应将在特定目标1中确定。