A novel promoter element, photoreceptor conserved element II, directs photoreceptor-specific expression of nocturnin in Xenopus laevis.
Nocturnin is a vertebrate circadian clock-regulated gene, and in Xenopus laevis its mRNA is specifically expressed in retinal photoreceptor cells. We have investigated the transcriptional regulatory mechanism that drives this precise spatial expression pattern of the nocturnin gene. A deletion series of the nocturnin 5'-flanking sequence driving the green fluorescence protein (GFP) reporter was used to generate transgenic Xenopus tadpoles. We found that a construct containing 2.6 kilobase pairs of 5'-flanking sequence targeted high level GFP reporter expression specifically to photoreceptor cells, in a pattern identical to endogenous nocturnin. This photoreceptor-specific expression pattern was maintained with several further deletions of 5'-upstream sequence, including a short 59-base pair fragment. Within this region of 59 base pairs, three perfect repeats of a novel protein binding site were identified by electrophoretic mobility shift assay. Competitions using varying oligonucleotide sequences demonstrated that the sequence required for protein binding is CAGACAGGCTATA, designated photoreceptor-conserved element II (PCE II). The protein complex that binds to this element is enriched in retinal extracts, and mutations of PCE II which fail to bind the protein complex also fail to direct GFP reporter expression to photoreceptors. These results indicate that the PCE II in the proximal promoter of the nocturnin gene is sufficient for driving the photoreceptor-specific expression of nocturnin.
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Department of Biology, National Science Foundation Center for Biological Timing, University of Virginia, Charlottesville, Virginia 22904-4328, USA.
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D000818:Animals; D001483:Base Sequence; D004247:DNA; D015342:DNA Probes; D005786:Gene Expression Regulation / Q000235:genetics*; D049452:Green Fluorescent Proteins; D007814:Larva / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D009687:Nuclear Proteins; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D014982:Xenopus laevis
D015342:DNA Probes; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C102804:nocturnin; D049452:Green Fluorescent Proteins; D004247:DNA