The modular nature of eukaryotic transcription factors has led to a picture of these regulatory proteins as composite molecules with discrete, autonomous units. Our studies of Ets-1, as a prototypic Ets protein, expands this view. DNA binding of Ets-1 is negatively regulated by a novel mechanism involving intramolecular interactions and conformational change. We plan to test our model of Ets-1 auto-inhibition and investigate mechanisms that reverse this negative regulation. I. To test the structural tenants of the model residues involved in packing of the proposed four helix bundle of the inhibitory module will be identified. A novel feature of auto-inhibition is the unfolding of an alpha helix within the inhibitory module. We propose that this structural transition could affect the global conformation and, thus, general function of Ets-1. This hypothesis will be tested by protease probing the Ets-1 structure. In addition, we will test for inhibitory effects on the Ets-1 activation domain which lies at some distance from the inhibitory module. II. We have proposed that the auto-inhibition of Ets-1 could be de-repressed by interaction with a partner protein. A candidate partner has been identified; in the Moloney murine leukemia virus enhancer, Ets-1 binds DNA cooperatively with the core binding factor (CBF). The mechanism of Ets- 1:CBF DNA binding cooperativity will be determined. Mechanisms that require protein:protein interactions and/or DNA conformation will be tested. The region(s) of Ets01 necessary for the cooperativity will be mapped and subjected to high resolution genetic and structural analysis. III. Phosphorylation and adaptor proteins will be investigated as possible pathways to modulate Ets-1 auto-inhibition. Two sources of phosphorylated Ets-1 will be exploited to test whether phosphorylation affects Ets I conformation, DNA binding and/or the CBF partnership. The pointed domain of Ets-1, which is highly-conserved among a subset of Ets-related proteins but has no known function, will be tested for activity as a protein interaction domain. Other do=mains characterized in this grant period will be used in additional screens for interacting proteins. In sum, our studies using the Ets family and the Moloney MLV enhancer should provide new insights into basic transcriptional control mechanisms. Novel strategies for regulating the function of transcription factors in pharmacological contexts should come from our detailed structural and mechanistic approaches. Ets-1 and other Ets proteins have been implicated in carcinogenesis as dominant oncogenes. This work is also related to the pathogenesis of retroviruses and their use in human gene therapy.
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