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KP Mitton, PK Swain, IJ Apel, A Swaroop; Functional Mapping Of Protein Interactions Involved In Regulation Of Gene Expression In Developing Retina: Nrl Molecular Interactions . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2439.
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Purpose: Cell-specific gene expression determines the fate of retinal progenitor cells as they differentiate into one of the six basic retinal neurons and glial cells. NRL is a transcription factor required for normal rod cell formation and is a component of the rhodopsin enhanceosome regulating the expression of rhodopsin. Protein interactions were mapped starting with different domains of NRL used as baits in yeast two-hybrid protein screening. Methods: Different domains of NRL were used as baits for multiple yeast two-hybrid screens of a bovine retina cDNA library, in addition to the previous reported screen using the NRL zipper domain (Mitton et al., 2000). Double positive clones were sequenced and evaluated by GST pull-down assay to confirm interactions. For rhodopsin promoter activation assay, CV-1 cells were co-transfected with expression constructs for NRL, interacting protein sequences, and the rhodopsin-promoter luciferase-reporter plasmid p130-Luc. Results: A second region of NRL, N-terminal to the DNA binding domain, was found to interact with CRX. Screening also identified an interactor with NRL that is a homolog of the novel mouse protein, Fiz-1. Unlike CRX, Fiz-1 is a ubiquitous signal transduction protein that can interact with the Flt-3 tyrosine kinase receptor in hematopoetic stem cells. GST pull-down assay using GST-NRL and 35S-bFiz confirmed NRL/bFiz interaction. Immunoblot analysis labeled a protein of 65 kDa in both bovine and human retina with the bFiz antiserum. Northern blotting showed human FIZ-1 transcripts in all human tissues examined and mouse Fiz-1 transcripts to be present in embryonic and postnatal mouse retina. In CV-1 cells, bFiz repressed NRL-mediated transactivation of the rhodopsin promoter in a dose dependent manner but did not affect CRX transactivation. Immunofluorescent staining of adult mouse retina with antibody to bFiz suggests ubiquitous protein distribution. Conclusion: Yeast two-hybrid trapping is useful for: 1) finding novel retinal-specific transcription factor and signal-transduction protein interactions, 2) mapping protein interactions to specific domains. Either of two regions of NRL, straddling the DNA binding domain, are sufficient for interaction with the CRX transcription factor. A novel and ubiquitous protein, Fiz1, also interacts with NRL and is present in the developing and adult mouse retina. The role of Fiz-1, and what cell-specific RTKs it may interact with in retinal tissue are not known.
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