Targeting DNA Secondary Structures for Bcl-2 Gene Regulation

描述（由申请人提供）：Bcl-2是一种膜蛋白，具有细胞凋亡抑制剂的功能。 bcl-2 的异常水平与许多人类疾病有关，包括癌症、神经系统疾病和心血管疾病。有效调节 bcl-2 表达为治疗这些疾病提供了希望。我们发现人类bcl-2基因含有富含GC的近端启动子区域，该区域可以利用重叠的富含鸟嘌呤的DNA序列形成两个稳定的分子内G-四链体DNA二级结构。这个富含 GC 的区域包含 WT1 蛋白的结合位点，该位点已被证明是 bcl-2 基因表达的负调节因子。我们最近开发了一种可以选择性结合 bcl-2 启动子 G-四链体结构的小分子化合物的筛选方法。有趣的是，这些化合物已被证明可以上调 bcl-2 基因转录。待测试的假设是，用小分子稳定 bcl-2 启动子 G-四链体二级结构可通过抑制负调节因子 WT1 蛋白的结合来上调 bcl-2 基因转录。 G-四链体 DNA 二级结构已被证明是人类 c-Myc 基因近端启动子区域的转录沉默元件，并且适合小分子药物靶向。 bcl-2 基因启动子区域中形成的 G-四链体可能与 c-Myc 启动子中的 G-四链体发挥相似的作用，因为它们的形成可用于调节基因转录。然而，bcl-2 启动子中 G-四链体结构的复杂性高于 c-Myc 启动子。富含 G 链区域重叠的两个可互换 G 四链体的存在可能对于 bcl-2 基因转录的精确调节很重要，因为每个 G 四链体可能与不同的蛋白质结合，导致不同的基因调节，其方式类似于噬菌体 lambda 中由相互作用的 Cro 和阻遏蛋白控制的遗传开关，其操纵子区域 (OR) 与彼此的启动子区域重叠，从而抑制彼此的转录。在本提案中，我们的目标是确定 bcl-2 启动子 G-四链体结构的生物学作用和分子结构。我们的主要方法，高场核磁共振波谱法，代表了在生理条件下测定 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特异性靶向bcl-2启动子G-四联体结构并有效调节bcl-2基因表达的小分子化合物。具体来说，我们计划 1) 确定两个可互换的 bcl-2 启动子 G-四链体在 bcl-2 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 bcl-2 基因的调控； 2) 确定两个可互换的 bcl-2 启动子 G-四链体及其与 G-四链体稳定化合物的复合物的结构。 公众健康相关性：bcl-2 水平异常与许多人类疾病有关，包括癌症、神经系统疾病和心血管疾病。有效调节 bcl-2 表达为治疗这些疾病提供了希望。拟议的研究代表了一种通过小分子药物调节 bcl-2 基因表达的新颖策略。

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.