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

描述(由申请人提供)：本提案的目标是证明酪氨酸羟化酶(TH)启动子区富含鸟嘌呤的(G-富G)片段可以形成多个分子内G-四链，具有不同的稳定性和构象，并且这些G-四链(GQs)在转录水平上差异地调节TH的表达。位于启动子区域的四个相邻的G-延伸形成的单个分子内GQ调控转录。一个关键但几乎没有答案的问题是，这种GQ是如何控制基因表达的。为了解决这一问题，我们将研究人类TH启动子的45个核苷酸片段，它包含7个Gs延伸段，位于转录起始点上游24个核苷酸的保守区内。推测这个富含G的片段可以采用多种稳定性和构象不同的GQ结构，是TH启动子中的一个关键调控元件。对45nT DNA及其部分片段的初步研究表明，存在多个GQ，它们的稳定性和构象存在差异。这项建议旨在通过利用单分子FRET和主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45nT片段中形成的GQ集合相关的构象的动态性和变异性。此外，针对真核生物中含量最丰富的单链DNA结合蛋白人类复制蛋白A(RPA)的展开活性，测试了这些结构的稳定性和生理相关性。此外，在整个TH启动子的背景下，将通过使用GFP和荧光素酶报告构建，使用点突变和缺失分析来确定45nt DNA的各种组件的功能性。了解TH基因表达的潜在机制很重要，因为异常的TH表达与许多精神问题有关，包括双相情感障碍、躁狂抑郁和精神分裂症。此外，TH表达神经元的缺失是帕金森病的一个主要因素。拟议的研究将在调控TH启动子活性的背景下建立45NT片段的结构-功能关系。破译TH启动子调控的分子机制不仅有助于更好地了解TH基因表达的调控，而且可能为治疗干预寻找新的靶点。 公共卫生相关性：酪氨酸羟基酶(TH)是多巴胺能神经元和其他组织中多巴胺生物合成的限速酶。酪氨酸羟化酶基因的异常表达与许多精神问题有关，包括双相情感障碍、躁狂抑郁、精神分裂症，它的缺失还与广泛的表型有关，从轻度的多巴反应性肌张力障碍(DRD)到严重的左旋多巴反应性帕金森综合症或进行性婴儿脑病的表型。了解TH启动子调控的分子机制不仅将有助于更好地了解TH基因表达的调控，还可能为多种神经疾病的治疗干预找到新的靶点，这些疾病使美国人口范围广泛。

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.