Understanding ETS transcription factors : from ordered domains to disordered sequences

ETS (E26 transformation specific) transcription factors play critical roles in regulating cellular growth, development and differentiation. They share a conserved ETS domain that interacts with specific DNA sequences and a subset of ETS proteins also contain a PNT domain responsible for protein partnerships. My research initially focused on the PNT domain of Drosophila Pointed-P2, with the goal of understanding the impact of phosphorylation on the activation of gene expression. Using a battery of NMR spectroscopic approaches, I demonstrated that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle. This helix must be displaced to allow docking of the PNT domain with the ERK2 MAP kinase Rolled, which in turn phosphorylates three N-terminal phosphoacceptor sites. The second part of my thesis focuses on three members of the ETS family called the ETV1/4/5 sub-group: ETV1 (Er81), ETV5 (Erm), and ETV4 (PEA3 (polyoma enhancer activator 3)). Using an extensive set of ETV4 deletion fragments, the DNA binding autoinhibitory sequences at both N- and C-terminal to the ETS domain were identified. Through detailed NMR spectroscopic studies, I confirmed that the inhibitory sequences are predominantly disordered and transiently interact with a coarsely defined surface on the ETS domain. This surface overlaps the DNA-recognition interface, thus indicating a steric mechanism of autoinhibition. Overall, my studies help define the molecular mechanisms underlying ETV1/4/5 factors autoinhibition, and may inspire new anti-cancer strategies. Finally, I also investigated the stability and dynamics of several uninhibited ETS domains, including ETV4, PU.1, Ets1, and ETV6. Using NMR spectroscopy, I determined the structure of the PU.1 ETS domain and identified an appended, C-terminal helix. Similar to Ets1 and ETV6, the DNA-recognition helix H3 of ETV4 and PU.1 are dynamic as evidenced by amide hydrogen exchange (HX). I also utilized molecular dynamics simulations to map the motions of the four ETS domains and identified several critical pathways that may impact their stabilities and possibly, the DNA-binding abilities. Overall, the data presented in my thesis will provide further understanding of the structure and regulation of the ETS transcription factors.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat