DNA binding regulation and functional specificity of the ETS protein TEL

Transcription factors are grouped into families defined by a highly conserved DNA binding domain, thus creating a paradox in transcriptional control. How do proteins with closely related DNA binding domains achieve promoter specificity? A variety of strategies exist that permit transcription factors to assume unique roles in gene regulation, including tissue-specific expression, divergence in the DNA binding domain, autoinhibition and cooperative protein partnerships. This dissertation explores the mechanisms that regulate DNA binding in the ETS protein TEL.;TEL polymerization via its PNT domain may play a role in defining TEL specificity and regulating TEL-mediated transcriptional repression. Using a TEL dimer as a model polymer, a combination of EMSAs, DNaseI protection assays and dissociation rate experiments demonstrated that TEL self-association facilitates cooperative binding to DNA. Notably, dimer cooperativity was observed on DNA duplexes containing binding sites with variable spacings and orientations, suggesting flexibility in the middle region of TEL. Transient transfection experiments revealed that a functional PNT domain was required for repression of a luciferase reporter driven by the endoA promoter, implying that a cooperative polymer is required for repression. Based on these findings, we propose that TEL bypasses autoinhibition by binding to DNA as a cooperative polymer, thus defining specificity by targeting TEL to promoters bearing multiple binding sites.;Quantitative DNA binding studies demonstrated that TEL DNA binding is regulated by autoinhibition. A deletion analysis mapped inhibitory sequences to residues 429-436 in the C-terminus, termed the C-terminal inhibitory domain (CID). Mutational analyses suggest that electrostatic interactions play an important role in stabilizing the inhibited conformation of TEL. Partial proteolysis coupled with circular dichroism spectroscopy suggest that disruption of electrostatic interactions results in a conformational change in the C-terminus of TEL that is not accompanied by a loss of helicity. Combining our biochemical data with the known structural data for the ETS domain of TEL, we propose that TEL displays a conformational equilibrium between two different structural states: One state is an inhibited conformation where the CID sterically blocks the DNA binding surface of TEL. The other state is an uninhibited conformation where the CID moves intact to facilitate DNA binding.