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Kecia L. Feathers, Cameron R. Strong, Sarah J. Garnai, Debra A. Thompson; Transcriptional Control of the Human RPE65 Promoter. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3343.
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In an effort to better understand the mechanisms involved in regulating gene expression necessary for the specialized functions of the RPE, the purpose of our studies was to identify the molecular interactions that regulate the promoter activity of RPE65, the human gene encoding the visual-cycle retinoid isomerase.
A series of reporter constructs was generated by cloning genomic sequence from the 5’-flanking region and first intron of human RPE65 (-2976 to +1275 relative to the TSS) upstream of the firefly-luciferase gene in pGL3. Each construct incorporated an ATG->TTG mutation of the RPE65-start codon. Expression constructs encoding MITF, OTX2, CRX, and NRL were generated in pcDNA3.1 using cDNAs amplified from human RPE/choroid-total RNA. Relative transcriptional activity was evaluated in transfected HEK293T and D407 cells in assays of firefly-luciferase activity normalized to the activity of a control Renilla luciferase construct.
Assays of luciferase activity in cells transfected with RPE65 reporter constructs showed that the maximal-transcriptional activity attributable to the upstream region was present within the sequence from -748 to +38. Constructs that included the complete intron 1 sequence exhibited significantly more activity than those containing only upstream sequence. However, the activity of constructs that included the first 315 bp of intron 1 was profoundly reduced, suggesting the presence of a silencer element in this region. Cotransfection of RPE65 reporter constructs with a cDNA encoding the transcription factor MITF that regulates TYR and BEST1 expression in the RPE had no positive effect on transcriptional activity. Similarly, the transcription factors CRX and Nrl involved in regulating gene expression in photoreceptor cells had little effect. However, cotransfection of a cDNA encoding the transcription factor OTX2 that is necessary for TYR expression significantly increased RPE65 reporter construct activity. No synergism was detected in assays of reporter constructs in cells that were cotransfected with the transcription factors in pairs.
Regulation of RPE65 promoter activity in transfected cells by OTX2, but not MITF, suggests that the mechanisms controlling the expression of the visual cycle genes are both unique, and overlapping, with those controlling other key aspects of RPE function. Further understanding of the molecular mechanisms that regulate RPE-gene expression will be important for future efforts to develop RPE-targeting vectors and cell-culture models.
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