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: 7858294
• 负责人: Tahir H Tahirov
• 金额: $ 26.2万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7858294
• 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控是通过DNA和有限数量的转录调控因子之间的复杂相互作用来实现的。其中代表性的范例是Runx1和Ets1蛋白合作DNA结合的复杂机制。人们普遍认为Runx1和Ets1的DNA结合活性被其DNA结合域两侧的序列自动抑制。Ets1和Runx1在与复合Ets-Runx dna结合元件协同结合时，通过直接物理相互作用发生相互激活。与Runx1不同的是，Ets1可以与另一个Ets1分子合作，在与间隔4bp的回文ets结合位点结合或与间隔较宽的ets结合位点结合时，抵消其自身的自抑制作用。本研究的总体假设是，参与两个或多个转录因子与基因调控区域合作结合的高阶蛋白质-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著不同。为了验证这一假设，提出了两个具体目标。在Specific Aim I中，将使用TCRa和Mo-MLV增强子的复合Ets-Runx结合元件来确定Runx1和Ets1协同DNA结合的结构基础，这代表了更高（约30倍）和更低（约10倍）的蛋白质协同结合程度，以及Ets和Runx结合位点之间的不同间距。在Specific Aim II中，将确定通过dna介导的同二聚化激活Ets1的结构基础：i)与间隔4bp的回文型ets结合位点的合作结合，II)与间隔广泛的ets结合位点的合作结合。x射线晶体学和多种方法（电泳迁移率转移测定、表面等离子体共振和原子力显微镜）将被应用于制定dna依赖性Runx1-Ets1和Ets1-Ets1合作的机制模型，这些模型将通过基因表达研究在体内进行测试。长期目标是通过高阶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复合物的结构既涉及正常细胞功能，也涉及导致癌症（特别是白血病和骨病）的疾病相关功能的改变，这将有助于开发新的、基于结构的治疗方法来对抗这些疾病。公共卫生相关性：在超过30%的人类白血病中，Runx1及其异二聚体伴侣Cbf2的基因被染色体易位、倒位和点突变破坏。Ets1在白血病和淋巴瘤中扩增和重排，并且在许多侵袭性和转移性实体肿瘤中观察到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。