Diversity in function: How a polydactyl zinc finger protein confers multiple functional outputs. James D Bullard¹, Ryan M Baxley², Jake M Traxler¹, Bianca N Mason¹, Pamela K Geyer^1,2. 1) Biochemistry Department, University Of Iowa; 2) Molecular & Cellular Biology Program, University of Iowa, Iowa City, IA.

   Zinc fingers (ZnFs) represent the most common DNA binding domain in metazoan transcription factors, with these proteins often carrying arrays of five or more ZnFs. It is unclear how individual ZnFs contribute to the function of such polydactyl transcription factors. While classically considered DNA binding motifs, ZnFs also direct protein-protein and RNA interactions, raising the possibility that ZnFs make regulatory contributions in addition to DNA association. To understand the role of individual ZnFs in a polydactyl DNA binding domain, we are studying Suppressor of Hairy-wing [Su(Hw)], a twelve ZnF DNA binding protein. This multifunctional transcription factor is required for gypsy insulator function and gene regulation in the ovary. Sequence comparisons demonstrate that the Su(Hw) ZnF domain is highly conserved, with each ZnF displaying 55% to 96% identity over 40 million years of evolution. We defined the in vitro DNA binding properties of bacterially produced full-length Su(Hw) and mutants that carry a disruption of a single ZnF. We found that eight of the twelve ZnFs contribute to binding, with four being essential. While the essential ZnFs are among the most conserved, the degree of conservation does not always correlate with a requirement for DNA binding. Interestingly, these studies uncovered distinct binding modes for Su(Hw), suggesting that ZnFs usage at genomic binding sites may impact the conformation of Su(Hw) and contribute to differential effects on transcriptional regulation. This postulate is being tested through in vivo studies of the ZnF mutants. To date, we have confirmed that the ZnFs essential for in vitro DNA binding are required for in vivo Su(Hw) function, as these mutants fail to rescue su(Hw)^null phenotypes. Investigations of the other ZnF mutants are underway. Together, these data will provide insights into how polydactyl transcription factors utilize different combinations of ZnFs to carry out multiple functions.