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S.C. Khani, E. Kasperek, J.E. Young; Interactions of a Compact Core Photoreceptor–Specific Enhancer/Promoter With Transcription Factors . Invest. Ophthalmol. Vis. Sci. 2006;47(13):807.
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© ARVO (1962-2015); The Authors (2016-present)
The conserved stretch immediately upstream of rhodopsin kinase (Rk) gene contains the key elements required for transcription in a spatially and temporally controlled pattern in both photoreceptor types. The purpose of the study is to identify and characterize the factors and sequences involved in photoreceptor–specific expression.
Transgenic mice carrying the crucial highly conserved 0.2–kb human Rk enhancer/promoter region upstream of the GFP reporter were examined for the relative levels of ocular GFP and Rk transcripts in Nrl and Crx positive or null background by immunostaining and Q–RT–PCR. Transient transfection were performed using various luciferase constructs carrying deletions and mutations in various cell types including retinoblastoma and RPE derived, and HEK293 cells. Cotransfections were used to test the effect of the activity of various transcription factors on the promoter activities.
The transcriptional activity of both the transgenic and endogenous Rk promoter is exquisitely sensitive to homeodomain factors and less so to the Nrl. Both the homeodomain binding site and the surrounding GC rich sequences contributed prominently to the activity of the enhancer. The activity of the construct was pronounced in retinoblastoma cells with only a small fraction of activity detected in RPE and kidney derived cells. Marked increase in promoter activity in 293 cells was observed upon contransfection with Otx2 and Crx, to a lesser extent by Sp1 and Sp4. This activity was only mildly enhanced by Nrl, but not Nr2e3.
Photoreceptor–specific activity depends primarily on the Otx–like homeodomain proteins interacting with Zn finger proteins primarily. Nrl only exerts a modest effect on the overall activity of the Rk. Identification of additional factors interacting with the region upstream of the transcription start sites is currently underway.
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