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。异常水平的bcl-2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的表达为这些疾病的治疗提供了希望。我们发现人类bcl-2基因含有一个富含gc的近端启动子区域，该区域可以通过重叠的富含鸟嘌呤的DNA序列形成两个稳定的分子内g -四重DNA二级结构。这个富含gc的区域包含一个WT1蛋白的结合位点，该蛋白已被证明是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 -四重结构的小分子化合物的筛选试验。有趣的是，这些化合物已被证明可以上调bcl-2基因的转录。待验证的假设是，小分子稳定bcl-2启动子g -四重二级结构通过抑制负调节因子WT1蛋白的结合而上调bcl-2基因的转录。g -四重体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，可用于小分子药物靶向。在bcl-2基因启动子区域形成的g -四重构象可能与c-Myc启动子区域的g -四重构象起着类似的作用，它们的形成可能起到调节基因转录的作用。然而，bcl-2启动子中g -四重体结构的复杂性高于c-Myc启动子。在富g链区域重叠的两个可互换的g -四重体的存在可能对bcl-2基因转录的精确调控很重要，因为每个g -四重体可能结合不同的蛋白质导致不同的基因调节，其方式类似于由相互作用的Cro和Repressor蛋白控制的噬菌体lambda中的遗传开关。它们的操作员区域（or）与彼此的启动子区域重叠，从而抑制彼此的转录。在这个提议中，我们旨在确定bcl-2启动子g -四重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，代表了在生理条件下测定DNA二级结构的主要工具，由于这种结构的结晶困难。我们的长期目标是利用基于结构的合理设计来开发专门针对bcl-2启动子g-四重结构的小分子化合物，并有效调节bcl-2基因的表达。具体而言，我们计划1)确定两个可互换的bcl-2启动子g -四联体在bcl-2基因调控中的功能意义，以及g -四联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 -四聚物及其与g -四聚物稳定化合物配合物的结构。

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.