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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038663-07
Application #
3295260
Study Section
Virology Study Section (VR)
Project Start
1987-07-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
7
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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De, Soumya; Okon, Mark; Graves, Barbara J et al. (2016) Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix. J Mol Biol 428:1515-30
Huang, Fu; Ramakrishnan, Saravanan; Pokhrel, Srijana et al. (2015) Interaction of the Jhd2 Histone H3 Lys-4 Demethylase with Chromatin Is Controlled by Histone H2A Surfaces and Restricted by H2B Ubiquitination. J Biol Chem 290:28760-77
De, Soumya; Chan, Anson C K; Coyne 3rd, H Jerome et al. (2014) Steric mechanism of auto-inhibitory regulation of specific and non-specific DNA binding by the ETS transcriptional repressor ETV6. J Mol Biol 426:1390-406
Clark, Kathleen A; Graves, Barbara J (2014) Dual views of SRF: a genomic exposure. Genes Dev 28:926-8
Desjardins, Geneviève; Meeker, Charles A; Bhachech, Niraja et al. (2014) Synergy of aromatic residues and phosphoserines within the intrinsically disordered DNA-binding inhibitory elements of the Ets-1 transcription factor. Proc Natl Acad Sci U S A 111:11019-24

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