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

描述（由申请人提供）：本提案的目的是证明酪氨酸羟化酶（TH）启动子区中富含鸟嘌呤（G-丰富）的片段可以形成具有不同稳定性和构象的多个分子内G-四链体，并且这些G-四链体（GQ）在转录水平上差异调节TH表达。已知由位于启动子区的四个相邻G-延伸形成的单个分子内GQ调节转录。一个关键但几乎没有答案的问题是，这些GQ的集合如何控制基因表达。为了解决这一问题，我们将研究一个45 nt的人类TH启动子片段，其中包含7个延伸的Gs，并位于一个保守的区域内，24 nt的转录起始位点的上游。据推测，该富含G的片段可以采用多种稳定性和构象不同的GQ结构，并且是TH启动子中的关键调控元件。对45 nt DNA及其片段子集的初步研究表明存在稳定性和构象不同的多个GQ。该提案旨在通过利用单分子FRET和批量荧光测量的独特能力，例如2-氨基嘌呤取代，结合其他生物物理和生物化学技术，来表征与在45 nt片段中形成的GQ的集合相关的构象的动态性和可变性。此外，这些结构的稳定性和生理相关性针对人复制蛋白A（RPA）（真核生物中最丰富的单链DNA结合蛋白）的解折叠活性进行测试。此外，在整个TH启动子的背景下，45 nt DNA的各种组分的功能性将通过使用GFP和荧光素酶报告构建体，使用点突变和缺失分析来确定。了解TH基因表达的潜在机制是重要的，因为异常的TH表达与许多精神疾病，包括双相情感障碍，躁狂抑郁症和精神分裂症有关。此外，TH表达神经元的损失是帕金森病的主要因素。这些研究将建立45 nt片段在调节TH启动子活性方面的结构-功能关系。破译TH启动子控制的分子机制不仅将导致更好地理解TH基因表达的调控，而且还可能确定新的治疗干预靶点。 公共卫生相关性：酪氨酸羟化酶（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包括双相情感障碍、躁狂抑郁症、精神分裂症在内的许多精神疾病相关，并且其缺陷也与从轻度端的TH缺陷型多巴反应性肌张力障碍（DRD）到重度端的左旋多巴无反应婴儿帕金森症或进行性婴儿脑病表型的广谱表型相关。了解TH启动子控制的分子机制不仅可以更好地了解TH基因表达的调控，而且还可以确定新的靶点，用于治疗各种各样的神经系统疾病，使广泛的美国人群受益。

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.