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K. M. Farjo, Y. Takahashi, G. Moiseyev, J.-X. Ma; Characterization of RDH10 Expression and Function in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3539. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinol Dehydrogenase 10 (RDH10) was recently found to be essential for retinoid metabolism during embryogenesis. In adults, RDH10 expression is largely restricted to the retinal pigment epithelium (RPE), where it is highly expressed. The goal of this study is to determine the cis-regulatory elements in the RDH10 gene responsible for RPE-specific expression and understand the role of RDH10 in RPE cell biology and retinoid cycling.
To study transcriptional regulation, the 5’-flanking region of the human RDH10 gene was cloned into the pGL3-basic firefly luciferase reporter plasmid. The pGL3-RDH10 promoter was co-transfected with the control renilla luciferase expression plasmid pRL-TK, and dual-luciferase reporter assays were used to determine promoter activity 24 h after transfection in hTERT-RPE1, D407, and HepG2 cells. Additionally, serial deletions of the pGL3-RDH10 promoter were designed based on phylogenetic footprinting and tested in the luciferase reporter assays as described above.
We found that RDH10 promoter activity is 2.5 to 18-fold higher in RPE cells than in other cell lines, confirming this promoter region drives robust expression in RPE cells. Serial deletions of the RDH10 promoter revealed that important cis-activating element(s) are present between -694 and -1458, relative to the transcription start site, and appear to be more important for promoter activity in RPE cells than in other cell types. No RPE-specific cis-activating elements are present between -352 and -694.
We have identified an activating cis-regulatory element between -694 and -1458 upstream of the RDH10 gene. We are currently testing more serial deletions between -694 and -1458 and performing electrophoretic mobility shift assays (EMSA) and DNAse footprinting to identify the exact transcription factor binding sites that are important for RPE-specific expression. Ongoing work also includes characterizing the enzymatic properties of RDH10 in vitro and in vivo using RDH10-deficient mice.
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