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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cell Development and Function Integrated Review Group (CDF)
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Tompkins, Laurie
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University of Utah
Internal Medicine/Medicine
Schools of Medicine
Salt Lake City
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Madison, Bethany J; Clark, Kathleen A; Bhachech, Niraja et al. (2018) Electrostatic repulsion causes anticooperative DNA binding between tumor suppressor ETS transcription factors and JUN-FOS at composite DNA sites. J Biol Chem 293:18624-18635
Currie, Simon L; Doane, Jedediah J; Evans, Kathryn S et al. (2017) ETV4 and AP1 Transcription Factors Form Multivalent Interactions with three Sites on the MED25 Activator-Interacting Domain. J Mol Biol 429:2975-2995
Currie, Simon L; Lau, Desmond K W; Doane, Jedediah J et al. (2017) Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5. Nucleic Acids Res 45:2223-2241
Desjardins, Geneviève; Okon, Mark; Graves, Barbara J et al. (2016) Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1. Biochemistry 55:4105-18
Kikani, Chintan K; Wu, Xiaoying; Paul, Litty et al. (2016) Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis. Elife 5:
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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