Auto-inhibition of DNA binding in the transcription factor Ets-: a mechanistic and structural study

The study of the ets family of proteins offers insight into the regulation of transcription factors. The transcriptional activity of ets family members is modulated by phosphorylation by several different signaling cascades, and interactions with other transcription factors. Characterization of several members of the family has identified auto-inhibition as a major component of this regulation. Here, I have used Ets-I as a model for understanding the mechanism of auto-inhibition. First, Ets-1 polypeptides lacking either one of two proposed inhibitory regions were characterized. Equilibrium and kinetic studies demonstrated that these two inhibitory regions cooperated functionally to destabilize protein-DNA complexes. Structural analysis demonstrated that the two regions were structurally coupled as well. These structural studies also demonstrated that loss of repression is accompanied by a conformational change in the inhibitory regions that is also transiently displayed when repressed forms bind to DNA. Secondary structural analysis identified inhibitory helices that interact with a helix in the ETS domain. A model is proposed in which this inhibitory module" regulates DNA binding affinity through conformational changes in the ETS domain. Functional assays were performed with full-length and a de-repressed form of Ets-1 in the yeast Saccharomyces cerevisiae. These experiments demonstrated that Ets-1 was a functional transcription factor in an organism with no known ets genes and that de-repression of the inhibitory module leads to increased transcriptional activity. Finally, genetic analysis of the C-terminal inhibitory helix identified individual amino acids involved in auto-inhibition. Possible in vivo modes of regulating the activity of the Ets-1 inhibitory module include phosphorylation and protein-protein interactions. These findings are crucial to understanding the function of Hts-1 auto-inhibition and provide a starting point of further investigations into Hts-1 regulation.