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A.S. Viczian, M. Theisen, M.E. Zuber; Regulation of the Vertebrate Optx2 Gene Promoter . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3119.
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© ARVO (1962-2015); The Authors (2016-present)
Despite its importance, relatively little is know about the molecular control of eye size. The homeobox containing transcription factor, Optx2 (Six6), has been shown to control eye size, however, the mechanism by which its expression is regulated has not yet been identified. Therefore, the purpose of this study is to identify important cis–acting elements and trans–acting factors necessary for Optx2 transcription.
We generated transgenic Xenopus laevis expressing GFP under the control of the human and frog Optx2 5' flanking region using the restriction enzyme mediated integration (REMI) method and compared their expression patterns. We also used deletion analysis to identify functionally important regions. Cross–species sequence comparisons were used to identify candidate cis–acting sites conserved among vertebrate Optx2 promoters. To determine if these sites were functionally relevant, we expressed the corresponding trans–acting factors in primitive ectoderm by RNA microinjection and assayed their effect on endogenous Optx2 expression using rt–PCR.
We observed only subtle differences between the expression patterns of GFP driven from the human and frog Optx2 promoters. Sequence analysis identified two large, highly homologous regions in vertebrate Optx2 promoters. These regions are approximately 125 and 182 base pairs in length and 59% and 83% identical between frog and human sequences, respectively. Deletion analysis demonstrated these regions are required for normal expression. At the most distal 5' region (about 1500bp upstream from the translation start site), Pax6 and neurogenin binding sites were identified. Preliminary data suggests that both appear to affect endogenous Optx2 expression.
Our results demonstrate remarkable functional conservation of the Optx2 5' flanking region between human and frog eyes. Cis–acting element conservation is demonstrated in the lengthy homologous regulatory regions found upstream of the Optx2 ATG start site. While Pax6 has been previously shown to induce Optx2 expression, initial studies suggest that neurogenin also affects Optx2 expression. Unknown elements also exist in the promoter, which will be analyzed in future studies.
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