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JE Young, S Finnegan, K Gross, D Higgins, SC Khani; Crx-Independent Expression of Rhodopsin Kinase (Rk) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1432.
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Purpose: The homeodomain transcription factor Crx has been shown to play an important role in the regulation of many photoreceptor-specific genes. The expression of some photoreceptor-specific genes, however, such as Rk, appear unaffected by the targeted disruption of the Crx gene. Our goal is to explore the mechanisms underlying Crx-independent photoreceptor-specific expression of Rk. Methods: RACE was used to amplify the previously uncharacterized leader sequence of Rk mRNA from C57BL/J mouse retina. The amplified cDNAs were cloned and sequenced and the promoter regions upstream of the mouse and human Rk gene were compared by BLAST algorithm. Conserved sequences within the 5' flanking region were fused upstream of the luciferase and LacZ reporter genes and used to transfect WERI retinoblastoma and dissociated human retinal cells in culture. The homeobox-binding site within the Rk promoter region was disrupted using site-directed mutagenesis in some of the constructs. The interaction of nuclear factors with the promoter sequences including the homeodomain-binding site was examined using gel retardation assay with nuclear extracts from normal BL/J and Crx-deficient mice. Results: Comparison of the upstream promoter region of both human and mouse rhodopsin kinase reveals ∼200-bp island of sequence homology containing a homeobox binding sequence CTAAT. Disruption of the homeobox sequence led to substantial decrease in reporter gene expression but did not abolish the activity of the promoter. No differences were observed in gel retardation assays when comparing the binding patterns of normal and Crx-/- mouse retinal nuclear proteins to the Rk homeobox-binding site. Conclusion: Our findings suggest that the mouse and human Rk genes share a highly conserved proximal promoter which is likely to be responsible for the appropriate developmental and photoreceptor cell-specific regulation of the gene. The proximal promoters in both genes contain a homeodomain-binding site whose disruption reduces but does not abolish promoter activity. The binding patterns of the promoter sequences to the nuclear proteins suggests a role for homeodomain transcription factor other than Crx in the regulation of Rk gene.
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