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.
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.
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