This proposal investigates the regulatory transcription factor Ets-1 as a model system for understanding the pathways to specificity within a gene family of DNA binding proteins. Ets-1 belongs to the ets family of transcription factors whose members share common DNA binding proteins. Ets-1 belong to the ets family of transcription factors whose members share common DNA binding properties. Our approaches are distinctive due to the extensive structural framework we have developed for Ets-1 and the rigorous quantitative analyses that we routinely perform. A major focus is the auto-inhibition phenomenon of Ets-1 in which DNA binding of the ETS domain is repressed by an intramolecular mechanism. Our recent experiments identified two sources of regulation that appear to act through the auto-inhibition mechanism. This proposal investigates both sources of regulation. 1. First, we will determine the mechanism by which Ca2+-dependent phosphorylation reinforces auto-inhibition, thus further repressing DNA binding. 2. A partnership between CBFalpha2 and Ets-1 counteracts the effects of auto-inhibition. Our goal is to test a model of this mechanism by obtaining structural data for the Ets-1: CBF ternary complex. We will also search for in vivo evidence for this partnership using chromatin immunoprecipitations. 3. The third specific aim focuses on the Pointed (PNT) domain of Ets-1. This region shows conservation within the ets family; however, a variety of functions have been proposed, including interactions with co-activators, co-repressors and self-association. We plan to directly test the oligomerization hypothesis and screen for interacting proteins. 4. Finally, in the fourth aim we develop a genetic approach to investigate the ets gene family within the C. elegans system. The 10 ets genes in C. elegans will be surveyed both for expression and by analysis of null phenotypes. Our initial focus is the function of the PNT domain within two C. elegans ets genes. Our initial focus is the function of the PNT domain within two C. elegans ets genes. Our work on the ets gene family is relevant to human disease. Ets-1 regulates gene expression of the Moloney murine leukemia virus and HIV-1, and we use transcriptional control elements from these viruses as model enhancers in our experiments. Second, the ets proteins, specifically the ETS and PNT domains, are involved in human cancers caused by chromosome translocations.
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