Structural Basis for Synergistic Gene Expression by Runx1 and Ets1 Proteins

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与有限数量的转录调节因子之间的复杂相互作用实现的。其中代表性的范例是由Runx 1和Ets 1蛋白协同DNA结合的详细机制。Runx 1和Ets 1的DNA结合活性被广泛认为是由其DNA结合结构域侧翼的序列自抑制的。Ets 1和Runx 1通过直接物理相互作用的相互激活发生在与复合Ets-Runx DNA结合元件合作结合时。与Runx 1不同，Ets 1可以与第二个Ets 1分子合作，以抵消其自身的自抑制作用，当结合到某些回文Ets结合位点时，Ets结合位点相隔4 bp，或者通过结合到广泛分离的Ets结合位点。本研究的总体假设假定，参与两个或更多个转录因子与基因的调控区的合作结合的高阶蛋白质-DNA复合物的类型根据转录因子的DNA结合位点之间的间隔而显著不同。为了检验这一假设，提出了两个具体目标。在特定目的I中，将使用TCR α和Mo-MLV增强子的复合Ets-Runx结合元件确定Runx 1和Ets 1协同DNA结合的结构基础，这代表了更高（>30倍）和更低（~10倍）程度的协同蛋白结合以及Ets和Runx结合位点之间不同间距的示例。在Specific Aim II中，将确定通过DNA介导的同源二聚化激活Ets 1的结构基础：i）与相隔4 bp的回文Ets结合位点协同结合，以及ii）与相隔很远的Ets结合位点协同结合。X射线晶体学和方法的组合（电泳迁移率变化测定，表面等离子体共振和原子力显微镜）将被应用于制定DNA依赖的Runx 1-Ets 1和Ets 1-Ets 1合作的机制模型，将在体内基因表达研究进行测试。长期目标是通过对高阶Runx和Ets复合物的X射线晶体学、生物物理学、生物化学和功能研究，建立Runx家族和Ets家族蛋白质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改变的疾病相关功能，有助于癌症，特别是白血病和骨骼疾病，将有助于开发新的基于结构的疗法来对抗这些疾病。公共卫生相关性：Runx 1及其异源二聚体伴侣Cbf 2的基因在超过30%的人类白血病中被染色体易位、倒位和点突变破坏。Ets 1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瘤中观察到Ets 1表达升高，包括乳腺癌、肺癌、结肠癌、胰腺癌和甲状腺癌。所提出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。

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.