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L. E. Kelly, H. M. El-Hodiri; Role of a Conserved Genomic Element in Retinal Homeobox (Rx) Gene Promoter Activity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4425. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal homeobox (Rx) gene is essential for eye development. It is expressed throughout retinal development from neural tube stages through retinal maturation. In the mature retina, Rx expression continues in all retinal progenitor cells (RPC) located in the ciliary marginal zone (CMZ) of larval Xenopus retina. Rx is also expressed in the photoreceptor layer (PRL) where it regulates gene transcription through photoreceptor conserved element one (PCE1). Identifying elements that regulate Rx transcription is important to understanding RPC and PR development. The purpose of these studies is to characterize the role of a 300bp ultra conserved element (UCE1) in regulating (or modulating) the activity of the Rx promoter.
DNA protein interactions were assayed in vitro by electrophoretic mobility shift assay using radiolabeled PCE-1 oligonucleotide probe and invitro translated proteins. Luciferase assays were performed by injecting synthetic mRNA effectors, UCE1-luciferase reporter, and an internal control into two-celled Xenopus embryos, cultured to various stages, lysed, and assayed for luciferase activity. Transgenics were generated by intra-cytosolic sperm injection, cultured to stage 41, and checked for GFP RNA expression. In situ hybridization was performed on paraffin embedded transgenic embryos sectioned at 8µm thickness, and detected by a DIG labeled riboprobe.
We identify an ultra conserved element, UCE1, 3’to the Rx coding region containing two putative PCE-1 sites, PCE1a and PCE1b. We have confirmed that Rx binds specifically to both PCE-1 sites in vitro. A UCE1 reporter construct increases in activity as development progresses, but is repressed by Rx. The Rx1a promoter is normally active in the PRL and a small portion of the CMZ. Addition of UCE1 to the Rx1a transgene driving GFP, results in additional GFP expression in the dorsal CMZ. Mutation of PCE1a results in reduction or absence of GFP expression in the CMZ and PRL. Similarly, mutation of the PCE1b reduces GFP expression in the CMZ and PRL. Mutating both PCE1 sites results in ectopic GFP.
Our data suggest that UCE1 is important for proper function of the Rx1a promoter and that Rx potentially regulates its own expression through UCE1.
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