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实现的。在代表性的范例中，有RUNX1和Ets1蛋白协同结合DNA的详细机制。人们普遍认为RUNX1和Ets1的DNA结合活性受到其DNA结合域两侧序列的自动抑制。Ets1和RUNX1通过直接物理相互作用在与复合ETS-RUNX DNA结合元件协同结合时相互激活。与RUNX1不同的是，Ets1可以与第二个Ets1分子合作，在与某些相隔4bp的回文ETS结合位点结合时，或通过与广泛分离的ETS结合位点结合来中和自己的自身抑制。这项研究的总体假设是，参与两个或更多转录因子与基因调节区协同结合的高序蛋白质-DNA复合体的类型明显不同，这取决于转录因子DNA结合位点之间的间距。为了验证这一假设，本文提出了两个具体目标。在特定目标I中，将使用TCRA和Mo-MLV增强剂的复合Ets-RUNX结合元件来确定RUNX1和Ets1协同结合DNA的结构基础，这代表了较高(~gt；30倍)和较低(~10)度的协同蛋白质结合，以及Ets和RUNX结合位点之间的不同间距。在特定的目标II中，将确定通过DNA介导的同源二聚化激活Ets1的结构基础：i)协同结合相隔4个碱基的回文Ets结合部位，以及ii)协同结合广泛分离的Ets结合部位。将应用X射线结晶学和多种方法(凝胶迁移率位移分析、表面等离子体共振和原子力显微镜)来建立DNA依赖的RUNX1-Ets1和Ets1-Ets1协同作用的机制模型，并将通过基因表达研究在体内进行测试。其长期目标是通过对高阶RUNX和ETS复合体的X射线结晶学、生物物理、生化和功能研究，建立一个全面的RUNX家族和ETS家族蛋白质转录调控的模型。RUNX和ETS复合体的结构既参与了正常的细胞功能，也参与了与癌症，特别是白血病和骨病相关的疾病相关功能的改变，这将有助于开发基于结构的新疗法来对抗这些疾病。与公共卫生相关：在超过30%的人类白血病中，RUNX1及其异二聚体Cbf2的基因受到染色体易位、倒位和点突变的干扰。Ets1在白血病和淋巴瘤中扩增和重排，并在许多侵袭性和转移性实体肿瘤中观察到高表达，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。

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.