Targeting DNA Secondary Structures for Bcl-2 Gene Regulation

针对 Bcl-2 基因调控的 DNA 二级结构

• 批准号: 8054269
• 负责人: DANZHOU YANG
• 金额: $ 29.46万
• 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: 2010-04-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054269
• 关键词: Address; Affect; Affinity; Apoptosis; BCL2 gene; Bacteriophage lambda; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Cardiovascular Diseases; Cells; Complex; Crystallization; DNA; DNA Sequence; Data; Disease; Drug Delivery Systems; Drug Design; Drug Interactions; EMSA; Effectiveness; Fluorescence Resonance Energy Transfer; G-Quartets; GC Rich Sequence; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Goals; Guanine; Human; Lead; Malignant Neoplasms; Mediating; Membrane Proteins; Methods; Molecular; Molecular Probes; Molecular Structure; NMR Spectroscopy; Operator Regions; Pharmaceutical Preparations; Physiological; Plasmids; Play; Promoter Regions; Protein Binding; Proteins; Regulation; Reporter; Repressor Proteins; Research; Role; Running; Screening procedure; Specificity; Structure; Techniques; Testing; Transcriptional Regulation; Transcriptional Silencer Elements; Up-Regulation; WT1 Protein; Work; base; c-myc Genes; design; drug structure; human disease; in vivo; inhibitor/antagonist; insight; nervous system disorder; novel; promoter; protein function; public health relevance; small molecule; small molecule libraries; tool; transcription factor

DESCRIPTION (provided by applicant): Bcl-2 is a membrane protein that functions as an inhibitor of cell apoptosis. Aberrant levels of bcl-2 are associated with many human diseases including cancer, neurological disorders, and cardiovascular diseases. Effective modulation of bcl-2 expression offers promise for the treatment of these diseases. We have found that the human bcl-2 gene contains a GC-rich proximal promoter region that can form two stable intramolecular G-quadruplex DNA secondary structures using overlapping guanine-rich DNA sequences. This GC-rich region contains a binding site of the WT1 protein which has been shown to be a negative regulator of the bcl-2 gene expression. We have recently developed a screening assay of small molecule compounds that can selectively bind the bcl-2 promoter G-quadruplex structures. Intriguingly, these compounds have been shown to upregulate the bcl-2 gene transcription. The hypothesis to be tested is that stabilization of the bcl-2 promoter G-quadruplex secondary structure(s) with small molecules upregulates bcl-2 gene transcription by inhibiting the binding of the negative regulator WT1 protein. A G-quadruplex DNA secondary structure has been demonstrated to be a transcriptional silencer element in the proximal promoter region of the human c-Myc gene and is amenable to small molecule drug targeting. The G-quadruplexes formed in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-Myc promoter in that their formation could serve to modulate gene transcription. However, the complexity of the G-quadruplex structures in the bcl-2 promoter is higher than is the case for the c-Myc promoter. The presence of two interchangeable G-quadruplexes overlapping in the region of the G-rich strand is likely to be important for the precise regulation of bcl-2 gene transcription, as each G-quadruplex may bind to different proteins leading to different gene modulation, in a manner analogous to the genetic switch in the bacteriophage lambda controlled by the interactive Cro and Repressor proteins, whose operator regions (ORs) overlap with each other's promoter regions and thereby inhibit each other's transcription. In this proposal we aim to determine the biological roles and molecular structures for the bcl-2 promoter G-quadruplex structures. Our primary approach, high-field NMR spectroscopy, represents a major tool for determination of DNA secondary structures under physiological conditions, due to the difficulty of crystallization of such structures. Our long-term goal is to use structure-based rational design to develop small molecule compounds that specifically target the bcl-2 promoter G-quadruplex structures and effectively modulate bcl-2 gene expression. Specifically, we plan to 1) determine the functional significance of the two interchangeable bcl-2 promoter G-quadruplexes in bcl-2 gene regulation and how binding of G-quadruplex-stabilizing compounds affects the regulation of the bcl-2 gene; and 2) to determine the structures of the two interchangeable bcl-2 promoter G-quadruplexes and their complexes with G-quadruplex-stabilizing compounds. PUBLIC HEALTH RELEVANCE: Aberrant levels of bcl-2 are associated with many human diseases including cancer, neurological disorders, and cardiovascular diseases. Effective modulation of bcl-2 expression offers promise for the treatment of these diseases. The proposed research represents a novel new strategy for modulating bcl-2 gene expression by small molecule drugs.

描述(申请人提供)：bcl2是一种膜蛋白，具有抑制细胞凋亡的功能。Bcl2的异常水平与许多人类疾病有关，包括癌症、神经疾病和心血管疾病。Bcl2基因表达的有效调控为这些疾病的治疗提供了希望。我们发现人类bcl2基因含有一个富含GC的近端启动子区域，该区域可以利用重叠的富鸟嘌呤DNA序列形成两个稳定的分子内G-四链DNA二级结构。这一富含GC的区域包含WT1蛋白的结合部位，已被证明是bcl2基因表达的负调节因子。我们最近开发了一种能够选择性结合bcl2启动子G-四链结构的小分子化合物的筛选方法。有趣的是，这些化合物已经被证明可以上调bcl2基因的转录。需要检验的假设是，bcl2启动子的G-四链二级结构(S)用小分子稳定，通过抑制负调控因子WT1蛋白的结合而上调bcl2基因的转录。已证实G-四链DNA二级结构是人c-Myc基因近端启动子区域的转录沉默元件，可用于小分子药物靶向。在bcl2基因启动子区域形成的G-四联体可能与c-Myc启动子中的G-四联体发挥类似的作用，因为它们的形成可以调节基因的转录。然而，bcl-2启动子中G-四链结构的复杂性高于c-Myc启动子。在富含G的单链区域存在两个可互换的G-四链体对于精确调控bcl2基因转录可能是重要的，因为每个G-四链体可能与不同的蛋白质结合，导致不同的基因调控，其方式类似于由相互作用的CRO和阻遏蛋白控制的基因开关，其操纵区(OR)与彼此的启动子区域重叠，从而抑制彼此的转录。在这项建议中，我们的目标是确定bcl2启动子G-四链结构的生物学作用和分子结构。我们的主要方法，高场核磁共振波谱，是在生理条件下确定DNA二级结构的主要工具，因为这种结构很难结晶。我们的长期目标是利用基于结构的合理设计来开发针对bcl2启动子G-四链结构的小分子化合物，并有效地调控bcl2基因的表达。具体地说，我们计划1)确定两个可互换的bcl2启动子G-四链体在bcl2基因调控中的功能意义，以及G-四链体稳定化合物的结合如何影响bcl2基因的调控；以及2)确定两个可互换的bcl2启动子G-四链体及其与G-四链体稳定化合物的复合体的结构。 公共卫生相关性：bcl2的异常水平与许多人类疾病有关，包括癌症、神经疾病和心血管疾病。Bcl2基因表达的有效调控为这些疾病的治疗提供了希望。本研究为小分子药物调控bcl2基因表达提供了一种新的策略。