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K.P. Mitton, J.M. Johnson, A. Swaroop; Fiz1: A Novel Zinc–finger Protein Binds the NRL Transcription Factor Using Internal Domains Different from its RTK–Binding Domains . Invest. Ophthalmol. Vis. Sci. 2004;45(13):714.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We recently discovered NRL–Fiz1 interaction (Hum Mol Genet, 2003) suggesting Fiz1 defines a novel zinc–finger repressor affecting retinal and non–retinal genes. We hypothesize that Fiz1’s repressive activity is mediated by interaction with Nrl in rods. Fiz1 can also bind to the developmental cell surface receptor Flt–3 using its terminal Znf–cluster domains (I and IV). To examine Fiz1’s molecular association with NRL we began to use yeast two–hybrid domain–deletion and peptide mapping. Methods: LexA–NRL–Zip (leucine–Zipper only) hybrid protein was expressed in yeast cells where it binds to Gal4AD–Fiz1 protein including domains II to IV. A Gal4AD–random peptide library was screened for short LexA–NRL–Zip binding peptides homologous to Fiz1. Direct interaction tests with hybrid activation–domain (AD)–Fiz1 deletions were examined for loss of interaction with LexA–NRL–Zip. Results: Random peptide library screening obtained 16–mer amino acid sequences with up to 80% homology to a portion of Fiz1 in domain II. Synthetic dsDNA was used to prepare the exact Fiz1 peptide sequence to confirm interaction using the yeast two–hybrid system. Initial deletion construction to remove domains I–III, show Fiz1 (dom IV) does not have strong interaction compared to Fiz1 (dom II–IV). Conclusions: Our current results suggest that Fiz1’s Flt–3–binding domains (I and IV) are separate from the NRL–binding domains that are domain II and possibly domain III. Flt3 uses multiple zinc–finger clusters for multiple protein interactions that allow it to interact with membrane RTKs and transcription factors.
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