Structural Basis for Synergistic Gene Expression by Runx1 and Ets1 Proteins

Runx1 和 Ets1 蛋白协同基因表达的结构基础

• 批准号: 7931247
• 负责人: Tahir H Tahirov
• 金额: $ 22.69万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931247
• 关键词: Affect; Area; Atomic Force Microscopy; Binding; Binding Sites; Biochemical; Biological Assay; Bone Diseases; Breast; Cell physiology; Chromosomal translocation; Colon; Complex; DNA; DNA Binding; DNA Binding Domain; Developmental Biology; Dimerization; Disease; Electrophoretic Mobility Shift Assay; Elements; Enhancers; Family; Gene Activation; Gene Expression; Genes; Goals; Homodimerization; Human; Investigation; Lead; Lung; Malignant Neoplasms; Malignant neoplasm of thyroid; Mediating; Methods; Modeling; Molecular; Mutate; Mutation; Nucleic Acid Regulatory Sequences; Organism; Pancreas; Pattern; Point Mutation; Prevention therapy; Protein Binding; Protein Family; Proteins; Regulation; Roentgen Rays; Solid Neoplasm; Stromelysin 1; Structure; Surface Plasmon Resonance; TP53 gene; Testing; Transcriptional Activation; Transcriptional Regulation; X-Ray Crystallography; base; comparative; fighting; human disease; in vivo; leukemia; leukemia/lymphoma; malignant stomach neoplasm; mutant; novel; promoter; public health relevance; success; transcription factor

DESCRIPTION (provided by applicant): Precise regulation of spatial and temporal patterns of gene expression is achieved by a complex interplay between DNA and a limited number of transcriptional regulatory factors. Among the representative paradigms are elaborate mechanisms of cooperative DNA binding by Runx1 and Ets1 proteins. The DNA binding activities of both Runx1 and Ets1 are widely believed to be autoinhibited by sequences flanking their DNA binding domains. Reciprocal activation of Ets1 and Runx1 by direct physical interaction occurs upon cooperative binding to composite Ets-Runx DNA-binding elements. Unlike Runx1, Ets1 can cooperate with a second Ets1 molecule to counteract its own autoinhibition when binding to certain palindromic Ets-binding sites separated by 4 bp or by binding to widely separated Ets-binding sites. The overall hypothesis of this study posits that the types of high-order protein-DNA complexes involved in cooperative binding of two or more transcription factors to the regulatory regions of a gene differ significantly depending on the spacing between the DNA binding sites for the transcription factors. To test this hypothesis, two specific aims are proposed. In Specific Aim I, the structural basis of cooperative DNA binding by Runx1 and Ets1 will be determined using composite Ets-Runx binding elements of TCRa and Mo-MLV enhancers, which represent examples of higher (>30 fold) and lower (~10) degrees of cooperative protein binding, as well as different spacing between Ets and Runx binding sites. In Specific Aim II, the structural basis of Ets1 activation by DNA-mediated homodimerization will be determined: i) for cooperative binding to palindromic Ets-binding sites separated by 4 bp, and ii) for cooperative binding to widely separated Ets-binding sites. X-ray crystallography and a combination of methods (Electrophoretic Mobility Shift Assays, Surface Plasmon Resonance and Atomic Force Microscopy) will be applied to formulate the mechanistic models of DNA-dependent Runx1-Ets1 and Ets1-Ets1 cooperation that will be tested in vivo by gene expression studies. The long-term goal is to build a comprehensive model of transcriptional regulation by the Runx family and the Ets family of proteins by means of X-ray crystallographic, biophysical, biochemical and functional studies of high-order Runx and Ets complexes. The structures of Runx and Ets complexes involved both in normal cellular function and altered disease-related function contributing to cancer, in particular leukemias, and bone diseases, will help to develop novel, structure-based therapies to fight these diseases. PUBLIC HEALTH RELEVANCE: The genes of Runx1 and its heterodimeric partner Cbf2 are disrupted by chromosomal translocations, inversions and point mutations in over 30% of human leukemias. Ets1 is amplified and rearranged in leukemia and lymphoma, and elevated Ets1 expression has been observed in many invasive and metastatic solid tumors, including breast, lung, colon, pancreatic and thyroid cancers. Success with the proposed structural studies ultimately will lead to the discovery of novel therapies for the prevention and treatment of leukemia and other cancers.

描述（由申请人提供）：基因表达的空间和时间模式的精确调节是通过DNA和有限数量的转录调节因子之间复杂的相互作用来实现的。代表性范例包括 Runx1 和 Ets1 蛋白协同 DNA 结合的复杂机制。人们普遍认为 Runx1 和 Ets1 的 DNA 结合活性受到其 DNA 结合域侧翼序列的自动抑制。 Ets1 和 Runx1 通过直接物理相互作用相互激活，发生在与复合 Ets-Runx DNA 结合元件协同结合时。与 Runx1 不同，当 Ets1 与某些相隔 4 bp 的回文 Ets 结合位点结合或与广泛分离的 Ets 结合位点结合时，Ets1 可以与第二个 Ets1 分子合作抵消其自身的自抑制。本研究的总体假设认为，参与两个或多个转录因子与基因调控区协同结合的高阶蛋白质-DNA 复合物的类型根据转录因子 DNA 结合位点之间的间距而显着不同。为了检验这一假设，提出了两个具体目标。在特定目标 I 中，Runx1 和 Ets1 协同 DNA 结合的结构基础将使用 TCRa 和 Mo-MLV 增强子的复合 Ets-Runx 结合元件来确定，这些元件代表较高（>30 倍）和较低（~10）的示例协同蛋白质结合的程度，以及 Ets 和 Runx 结合位点之间的不同间距。在特定目标 II 中，将确定 DNA 介导的同二聚化 Ets1 激活的结构基础：i) 与相隔 4 bp 的回文 Ets 结合位点协同结合，ii) 与广泛分离的 Ets 结合位点协同结合。 X射线晶体学和多种方法（电泳迁移率变化测定、表面等离子共振和原子力显微镜）的组合将用于制定DNA依赖的Runx1-Ets1和Ets1-Ets1合作的机制模型，该模型将在体内进行测试基因表达研究。长期目标是通过对高阶Runx和Ets复合物的X射线晶体学、生物物理、生化和功能研究，建立Runx家族和Ets家族蛋白转录调控的综合模型。 Runx 和 Ets 复合物的结构涉及正常细胞功能和导致癌症（特别是白血病）和骨病的疾病相关功能改变，将有助于开发新的、基于结构的疗法来对抗这些疾病。公共健康相关性：在超过 30% 的人类白血病中，Runx1 及其异二聚体伙伴 Cbf2 的基因因染色体易位、倒位和点突变而受到破坏。 Ets1 在白血病和淋巴瘤中被扩增和重排，并且在许多侵袭性和转移性实体瘤中观察到 Ets1 表达升高，包括乳腺癌、肺癌、结肠癌、胰腺癌和甲状腺癌。拟议的结构研究的成功最终将导致发现预防和治疗白血病和其他癌症的新疗法。