Identification of cis–elements contributing to cone photoreceptor–specific expression

Abstract

Abstract: : Purpose: A 215–bp proximal promoter fragment of mouse cone arrestin (mCAR) containing a TATA box, a TATA like element, four cone–rod homeobox (CRX)–binding sites and one Sp1 site is sufficient to drive cone photoreceptor– and pineal–specific expression. This study is to understand the transcriptional regulation underlying cone–specific expression of the mCAR promoter. The contribution of each cis–element to the cone–specific expression was examined. Methods: The 5’ deletion and single cis–element–mutated mCAR promoter fragments were created by PCR and site–directed mutagenesis and subcloned into both pRL–Null and pEGFP vectors. COS–7 and Weri–Rb–1 retinoblastoma cells were transient transfected by different promoter–luciferase reporter constructs with or without a bovine CRX expression construct. Luciferase assays were examined for promoter activities. Transgenic Xenopus laevis tadpoles expressing mutant promoter–EGFP reporter constructs were generated, screened for EGFP expression and genotyped by genomic PCR with tail DNA. Cryosections of tadpole heads were used to examine EGFP expression and distribution in the retina. Results: Transient transfection assay results suggested: 1) no significant enhancer in further 5’ upstream region nor the first intron of the mCAR gene, and 2) the mutation of each cis–element has different levels of effect on promoter activity. Transgenic tadpoles revealed: 1) with two 5’ deletion constructs (181–bp or 147–bp), EGFP expression was also observed in tail muscle, heart and brain; 2) with either a mutated TATA box or a mutated TATA–like element, no EGFP expression in retina and was weaker in pineal, brain, tail muscle and heart; 3) with a single mutation of three of the four CRX–binding sites EGFP expression was retained in eyes but also in muscle and brain; 4) when the most proximal CRX–binding site was mutated, EGFP expression was not detectable; 5) with the mutated Sp1 site, EGFP expression pattern was random. Conclusions: Our results demonstrate that the TATA box, TATA–like element and the most proximal CRX–binding site are critical for the transcriptional targeting of mCAR promoter. The Sp1 site is suggested to facilitate the cone–specific expression of mCAR. The other three CRX–binding sites may serve as repressors in tissues other than the retina and the pineal gland.

Keywords: photoreceptors • gene/expression • transcription factors