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Y. Pan, S. Nekkalapudi, L. E. Kelly, H. M. El-Hodiri; Combinatorial Regulation of Photoreceptor Gene Transcription by the Retinal Homeobox (Rx) Gene Product and Other Retinal Transcription Factors. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2920.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal homeobox (Rx) gene product is essential for eye development. However little is known about the molecular function of Rx. No target genes have been identified and it is not known if Rx cooperates with other transcription factors to regulate transcription. It has been shown that in vitro Rx binds to PCE-1 site, which is a highly conserved element in the promoter region of photoreceptor related genes including rhodopsin gene. The rhodopsin promoter contains four highly conserved regulatory elements, PCE-1, BAT (Ret2), NRE (Ret3), and Ret4. The purpose of this study is to confirm that Rx binds to PCE-1 site in vivo, and to characterize potential functional interactions between Rx and other factors involved in regulation of rhodopsin expression.
Xenopus opsin promoter - luciferase (XOP-Luc) DNA and RNAs encoding transcriptional regulators were microinjected into two-cell Xenopus laevis embryos. Luciferase activity was measured in lysates prepared from injected mid-gastrula embryos. Mobility shift assays (EMSA) were performed using in vitro translated proteins, radiolabeled PCE-1 probe, and oligonucleotide competitors. Expression patterns were analyzed by in situ hybridization using whole or sectioned embryos and digoxigenin-labeled antisense riboprobes. Chromatin immunoprecipitation (ChIP) was performed using eyes isolated from Xenopus embryos over-expressing myc-tagged Rx and anti-myc (9E10) or control antibody. The presence of rhodopsin and red cone opsin promoters in the ChIP products were confirmed by quantitative PCR using primers flanking the PCE-1 site of rhodopsin and red cone opsin promoters.
Rx is expressed in maturing photoreceptors where genes regulated by PCE-1-containing promoters are expressed. Chromatin IP using stage 41 Xenopus tadpole eyes suggests that Rx binds to the rhodopsin and red cone opsin promoters in vivo. Rx can cooperate with otx5b and XLMaf (the Xenopus analogs of Crx and Nrl) to activate a luciferase reporter gene containing the Xenopus opsin promoter. We also find that additional retinal transcription factors, products of the Rx-L, Vax, and Vsx genes, bind the PCE-1 site in vitro and, therefore, may also be involved in regulating opsin promoter activity.
Our data demonstrates that opsin genes are direct targets of Rx and Rx can co-operate with other transcription factors to regulate rhodopsin gene expression and photoreceptor development.
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