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Qiong Qin, Konstantin Petrukhin; NR2E3, A Retina-specific Nuclear Receptor, Directly Regulates Transcription Of The Antioxidant Enzyme PON1. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6473.
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Oxidative stress plays significant role in pathogenesis of AMD. Paraoxonase 1 (PON1) is a protective antioxidant enzyme responsible, among other activities, for detoxification of peroxidized lipids. PON1 was found to be differentially expressed in the rd7 retina, implying that it may be directly regulated by retina-specific nuclear receptor NR2E3. In this study we provide evidence that PON1 is a direct target for NR2E3.
Two-dimensional electrophoretic mobility shift assay (2D-EMSA) was used to identify DNA fragments containing NR2E3 response elements in the proximity of potential target genes. In silico analysis was employed for pinpointing potential NR2E3 response elements (REs) in the DNA fragment trapped from the BAC clone containing PON1. Conventional EMSA with double stranded synthetic oligonucleotide containing the putative PON1RE was used to establish direct NR2E3 interaction with the potential response element. In order to confirm that PON1 RE identified in 2D-EMSA is functional in a cellular context, a reporter construct containing the trapped DNA fragment attached to a minimal promoter was used to assess the effect of NR2E3 on a reporter gene transcription.
Analysis of the trapped 343-b.p. fragment located upstream of the classic 2.9 kb PON1 promoter revealed a response element combining two established Knix2 and DR1 hexameric half sites. Conventional EMSA confirmed direct NR2E3 interaction with the predicted RE. Transient transfections of the pGL4.10 reporter vector containing the 343-b.p. fragment confirmed that NR2E3 dose-dependently repressed the transcription of the reporter gene indicating that RE identified in 2D-EMSA is functional.
NR2E3 seems to directly regulate PON1 transcription. NR2E3-mediated upregulation of the protective enzyme, PON1, with small molecule NR2E3 modulators may potentially be used as a therapeutic approach for treatment of dry AMD.
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