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，作为细胞凋亡的抑制剂发挥作用。bcl-2的异常水平与许多人类疾病相关，包括癌症、神经系统疾病和心血管疾病。bcl-2表达的有效调节为这些疾病的治疗提供了希望。我们已经发现，人类bcl-2基因包含一个富含GC的近端启动子区域，该区域可以使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。该富含GC的区域含有WT 1蛋白的结合位点，该结合位点已被证明是bcl-2基因表达的负调节剂。我们最近开发了一种筛选分析的小分子化合物，可以选择性地结合bcl-2启动子G-四链体结构。有趣的是，这些化合物已被证明上调bcl-2基因转录。待检验的假设是，用小分子稳定bcl-2启动子G-四链体二级结构通过抑制负调节因子WT 1蛋白的结合而上调bcl-2基因转录。已证明G-四链体DNA二级结构是人c-Myc基因近端启动子区域中的转录沉默元件，并且适合于小分子药物靶向。在bcl-2基因启动子区形成的G-四链体可能发挥与c-Myc启动子中的G-四链体类似的作用，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启动子的情况。在富含G链的区域中重叠的两个可互换的G-四链体的存在对于bcl-2基因转录的精确调节可能是重要的，因为每个G-四链体可以以类似于由相互作用的Cro和阻遏蛋白控制的λ噬菌体中的遗传开关的方式结合到导致不同基因调节的不同蛋白，其操纵子区（OR）与彼此的启动子区重叠，从而抑制彼此的转录。在这个提议中，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NMR光谱，是一个主要的工具，用于确定DNA二级结构在生理条件下，由于结晶的困难，这样的结构。我们的长期目标是使用基于结构的合理设计来开发特异性靶向bcl-2启动子G-quadruplex结构并有效调节bcl-2基因表达的小分子化合物。具体而言，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;以及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。 公共卫生相关性：bcl-2的异常水平与许多人类疾病相关，包括癌症、神经系统疾病和心血管疾病。bcl-2表达的有效调节为这些疾病的治疗提供了希望。该研究为小分子药物调控bcl-2基因表达提供了一种新的策略。

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.