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Jennifer Bosse, Reyna Martinez-De Luna, Heithem M El-Hodiri; Genetic Analysis of Retinal Homeobox Enhancer UCE2. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1491.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal homeobox (Rx) gene is necessary for maintaining retinal stem cells (RSCs) and retinal progenitor cells (RPCs) in Xenopus but regulation of Rx transcription is not well understood. This study tests the hypothesis that Rx expression is regulated in RSCs and RPCs by protein binding sites within discrete regions of Rx enhancer UCE2.
Ten base pair (bp) pieces from UCE2 were sequentially removed and the remainder cloned into plasmids containing the Rx promoter region and GFP (reporter). The cloning was repeated using plasmids that contained bps 51-250 of UCE2. Transgenes, generated from the cloned plasmids, were introduced into Xenopus laevis eggs through intra-cytosolic sperm injection. The transgenic animals, identified by GFP expression, were cultured to stage 42, paraffin-embedded, sectioned and probed for GFP expression by in situ hybridization using a DIG-labeled antisense probe.<br /> The TCF binding sites of the TOPFLASH luciferase plasmid were replaced with bps 200-250 of UCE2. HEK293T cells were transfected with this new construct along with GFP, a promoterless Renilla luciferase reporter and either a control plasmid, pSC2, or a dominant negative TCF construct. LiCl was added to the media of transfected cells for 24 hours to activate Wnt signaling and cell lysates were used to perform a dual luciferase assay.
Removal of bps 11-40 eliminated or reduced reporter transcription in the RSCs, while removal of bps 1-10 had no effect on reporter transcription in the CMZ. In the context of having the additional UCE2 bps 51-250, removal of bps 1-20 had no effect on reporter transcription in the CMZ. There was an increased luciferase expression in response to LiCl in cells transfected with TOPFLASH containing bps 200-250 of UCE2. While cells also transfected with dominant negative TCF had decreased luciferase expression when compared to positive control.
Our in situ hybridization data suggests that cis-acting element(s) within bps 21-40 of UCE2 are necessary for transgene expression in the RSCs of the CMZ. The element(s) could be transcription factor binding site(s) which, when disrupted or removed, alters transgene expression in the CMZ. The partial reduction in transgene expression accompanying the deletion of bps 11-20 may represent a partial deletion in one of the cis-acting elements.<br /> Our cell culture data suggests that transcription regulated by the bps 200-250 of UCE2 may be modulated by Wnt signaling.
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