The broad objective of this proposal is to biochemically characterize the ets1 and ets2 gene products as eukaryotic transcription factors. Ets l and Ets2 are members of the newly-described family of DNA binding transcription factors. The proteins in the ets family are characterized by a highly conserved 85-amino acid domain. The v-ets gene product, the first ets protein described, is encoded by the transforming oncogene of the avian retrovirus E26. Our laboratory was instrumental in identifying the conserved region of ets family members as a DNA binding domain and calling it the ETS domain. This domain shows no strong similarity to DNA binding domains of other gene families suggesting that it encodes a novel structural motif.
Our specific aims focus on the important issue of specificity in the ets family. How are different members of the family targeted to function in different regulatory pathways? At least part of the answer to this question will lie in the sequence-specificity of DNA binding, thus our immediate goal is to continue our characterization of the specific DNA binding of the Ets1 ETS domain. Our chemical and genetic approaches have already provided a preliminary picture of the DNA contacts made by the ETS domain. We propose to continue these approaches in order to completely define the DNA binding site of Ets1. Next, a structural characterization of the ETS domain is planned. We have already developed a high level bacterial expression system for the Ets1 ETS domain that will expedite these studies. To complement the structural studies, we will perform a genetic screen to identify Ets1 mutations affecting DNA binding. These genetic and biochemical approaches will lead to a model of the mechanism of DNA binding by the ETS domain. We also are exploring other potential sites of regulation of the ets family members using Ets 1 and Ets2 as model ETS domain proteins. Experiments are proposed to investigate the transcription function of Ets1 and Ets2 and their competence to interact with other proteins. A specific goal is to map by deletion mutagenesis the domains of the Ets1 and Ets2 that mediate these other functions. These initial aims will set the stage for more mechanistic studies. We discovered the DNA binding activity of the ets family in the context of our search for transcriptional machinery utilized by the the Moloney murine leukemia virus (MLV) in T lymphocytes. The binding sites of Ets1 and Ets2 map to the activator elements that have been shown to modulate the disease specificity of MLV. We have shown that the expression of Ets l and Ets2 in T cells correlates with the activity of the Moloney LTR. Our planned studies will test more directly for the ability of Ets l and Ets2 to activate the MLV enhancer. For example, we plan to investigate the interaction of these ets proteins with the Moloney enhancer core binding protein CBP that also binds to a determinant of MLV disease specificity. In conclusion, our molecular studies will exploit the ets family as a powerful system to elucidate rules for eukaryotic transcriptional regulation and viral pathogenesis.
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