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A Conserved G-rich Region within Human TH Promoter Regulates Its Activity

人类 TH 启动子内的保守富含 G 区域调节其活性

基本信息

批准号：
8180612
负责人：
Soumitra Basu
金额：
$ 28.08万
依托单位：
KENT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-18 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8180612
关键词：
2-Aminopurine Address Adopted Anabolism Attention Base Pairing Binding Biochemical Bipolar Disorder Catecholamines Complex DNA DNA Sequence Data Disease Dopa-Responsive Dystonia Dopamine Encephalopathies Enzymes Eukaryota Fluorescence G-Quartets Gene Expression Genes Genetic Transcription Genomics Goals Growth Factor Gene Guanine Human Human Activities Human Genome Lead Levodopa Link Luciferases Manic Measurement Mental Depression Molecular Molecular Conformation Mutation Analysis Neurons Oligonucleotides Oncogenes Parkinson Disease Parkinsonian Disorders Phenotype Physiological Play Point Mutation Population Promoter Regions Property Proteins Regulation Regulatory Element Reporter Role SS DNA BP Schizophrenia Stretching Structure Structure-Activity Relationship Techniques Testing Therapeutic Intervention Tissues Transcription Initiation Site Transcriptional Regulation Tyrosine 3-Monooxygenase base deletion analysis dopaminergic neuron infancy interest nervous system disorder non-oncogenic promoter replication factor A single-molecule FRET

项目摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to demonstrate that a Guanine-rich (G-rich) segment in the promoter region of Tyrosine Hydroxylase (TH) can form multiple intramolecular G-Quadruplexes, with varying stability and conformation, and that these G-quadruplexes (GQs) differentially regulate TH expression at the transcription level. Single intramolecular GQ formed by four neighboring G-stretches located in promoter regions are known to regulate transcription. A critical but barely answered question is how ensemble of such GQs controls gene expression. To address this we will study a 45 nt segment of human TH promoter that contains seven stretches of Gs and is located within a conserved region, 24 nt upstream of the transcription start site. It is hypothesized that this G-rich segment can adopt multiple GQ structures that vary in stability and conformation and is a key regulatory element in TH promoter. Preliminary studies on the 45 nt DNA and a subset of its fragments suggest the presence of multiple GQs that vary in stability and conformation. This proposal aims to characterize the dynamicity and variability in conformations associated with the ensemble of GQs formed in the 45 nt segment by utilizing the unique capabilities of single molecule FRET and bulk fluorescence measurements, e.g. 2-aminopurine substitution, in combination with other biophysical and biochemical techniques. Furthermore, the stability and physiological relevance of these structures are tested against unfolding activity of human Replication Protein A (RPA), the most abundant single strand DNA binding protein in eukaryotes. In addition, the functionality of the various components of the 45 nt DNA in the context of the entire TH promoter will be determined by using GFP and luciferase reporter constructs using point mutation and deletion analysis. Understanding the underlying mechanism of TH gene expression is important because aberrant TH expression is linked to many psychiatric problems including bipolar disorder, manic depression, and schizophrenia. Additionally, loss of TH expressing neurons is a major factor in Parkinson's disease. The proposed studies will establish the structure-function relationship of the 45 nt segment in the context of regulation of TH promoter activity. Deciphering the molecular mechanism of TH promoter control will not only lead to better understanding of regulation of TH gene expression but may also identify new targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Tyrosine Hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis in the dopaminergic neurons and other tissues. Aberrant expression of Tyrosine Hydroxylase gene is linked to many psychiatric problems including bipolar disorder, manic depression, schizophrenia, and its deficiency is also associated with a broad spectrum of phenotype ranging from TH-deficient dopa-responsive dystonia (DRD) at the mild end to a levodopa-unresponsive infantile parkinsonism or progressive infantile encephalopathy phenotype at the severe end. Understanding of the molecular mechanism of TH promoter control will not only lead to better understanding of regulation of TH gene expression but may also identify new targets for therapeutic intervention of a wide variety of neurological disorders benefitting a wide spectrum of US population.

描述（由申请人提供）：本提案的目标是证明酪氨酸羟化酶（TH）启动子区域的富鸟嘌呤（G-rich）片段可以形成多个具有不同稳定性和构象的分子内g -四plex，并且这些g -四plex （GQs）在转录水平上差异调节TH的表达。单个分子内GQ由位于启动子区域的四个相邻g -stretch组成，已知可调节转录。一个关键但几乎没有答案的问题是，这些智商的集合是如何控制基因表达的。为了解决这个问题，我们将研究人类TH启动子的45 nt片段，该片段包含7个Gs片段，位于转录起始位点上游24 nt的保守区域内。假设这个富含g的片段可以采用多种稳定性和构象不同的GQ结构，是TH启动子的关键调控元件。对45nt DNA及其片段子集的初步研究表明，存在多种gq，其稳定性和构象各不相同。本提案旨在通过利用单分子FRET和体荧光测量的独特能力，结合其他生物物理和生化技术，表征与45 nt段形成的gq集合相关的构象的动态和可变性。例如2-氨基嘌呤取代。此外，这些结构的稳定性和生理相关性通过真核生物中最丰富的单链DNA结合蛋白人类复制蛋白A （RPA）的展开活性进行了测试。此外，在整个TH启动子的背景下，45nt DNA的各种组成部分的功能将通过使用GFP和荧光素酶报告基因结构以及点突变和缺失分析来确定。了解TH基因表达的潜在机制非常重要，因为异常的TH表达与许多精神疾病有关，包括双相情感障碍、躁狂抑郁症和精神分裂症。此外，TH表达神经元的缺失是帕金森病的一个主要因素。这些研究将在TH启动子活性调控的背景下建立45nt片段的结构-功能关系。破解TH启动子调控的分子机制不仅有助于更好地理解TH基因表达的调控，而且可能为治疗干预找到新的靶点。