Autoinhibition and DNA-Binding Cooperativity in the Transcription Factor ETS1
Samorodnitsky, Daniel Gilbert
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Sequence specific transcription factors are a class of proteins that bind to specific DNA sequences in regulatory regions, like promoters and enhancers, and affect the transcriptional activity of associated genes. Some transcription factor regulate their own activity through “autoinhibition”, a phenomenon where a protein blocks its own function until certain conditions are met, autoinhibition is relieved, and the protein is activated. Relief of autoinhibition is often mediated by a change in secondary structure induced by interaction with another protein. The metazoan transcription factor ETS1 has been shown to autoinhibit, by blocking its ability to bind to DNA. Additionally, it has been shown to form both homodimeric and heterodimeric complexes. However, how autoinhibition and DNA-binding are related has not been clearly delineated. In this study I have investigated the relationships between DNA-binding, homodimerization, and autoinhibition in both full-length ETS1 and the splice variant ETS1-p42. It is split into two chapters, with one chapter dedicated to each protein. In the first chapter, I focus on full-length ETS1, studying the formation of homodimers on DNA containing a dimer binding site and DNA that does not. This includes studying the ability of proteins to form stable complexes with various DNA sequences, the ability of ETS1 to interact with DNA without forming complexes, and the varying degrees that different DNA sequences induce homodimer formation. Additional experiments address the link between non-specific DNA interrogation and homodimer formation. From there, I focus on elucidating a new role for autoinhibition in ETS1, and the specific regions of the protein involved in this new function. The second chapter focuses on the splice variant, ETS1-p42. This chapter explains previously published reporter assays showing that despite binding as a monomer, this protein requires two EBSs to drive luciferase expression. I do this by showing both DNA-binding cooperativity and simultaneous binding at both EBSs by ETS1-p42, indicating that normal ETS1-p42 function requires dimeric DNA binding. Furthermore, I show that residues in ETS1-p51 that mediate autoinhibition are the same residues that mediate cooperativity in ETS1-p42. This chapter corroborates and furthers findings from chapter 2 and from previous work on the ETS protein SPI1 that showed that an uninhibited ETS domain semi-stably self-associates.