Nicoletti A, Kawase K, Thompson D A
Department of Ophthalmology, University of Michigan Medical School, Ann Arbor 48105-0714, USA.
Invest Ophthalmol Vis Sci. 1998 Mar;39(3):637-44.
To identify the functional promoter region and cis-acting elements that regulate the expression of RPE65, the retinal pigment epithelium (RPE)-specific gene responsible for certain forms of autosomal recessive childhood-onset severe retinal dystrophy.
A human genomic DNA clone containing the 5'-flanking region of RPE65 was isolated and, 4.0 kb proximal to the transcription start site, was sequenced and analyzed for the presence of transcription factor-binding sites. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the upstream sequence in the human RPE cell lines ARPE19 and D407, as well as in the SK-Mel-28 and HeLa cell lines. Specific DNA protein-binding sites present in the 340 bp upstream of the transcription start site were identified by DNase I footprint analysis.
Sequence analysis places the polymorphic marker, D1S2803, within the RPE65 upstream region and identifies a number of sequences homologous to the gene encoding the cellular retinaldehyde-binding protein. Functional analysis indicates that basal promoter activity is conferred by the sequence from -83 to +39 and is approximately equivalent in all cell lines tested, with no other control elements detected in 3.6 kb of the upstream sequence. At least eight protected regions are identified in DNase I footprint assays, including sequences corresponding to the predicted TATA box, AP-4, and nuclear factor-1 DNA protein-binding sites.
These findings localize the basal promoter activity of RPE65, identify potential cis-acting elements that act as positive regulators of gene expression, and suggest that additional regulatory elements are likely to be involved in restricting gene expression to the retinal pigment epithelium. Identification of promoter elements and genetic markers in the upstream sequence will enable the screening of patients with retinal degeneration for possible mutations that affect RPE65 expression.
鉴定调节RPE65表达的功能启动子区域和顺式作用元件，RPE65是视网膜色素上皮（RPE）特异性基因，与某些常染色体隐性遗传性儿童期严重视网膜营养不良的发病有关。
分离出包含RPE65 5'侧翼区的人类基因组DNA克隆，对转录起始位点近端4.0 kb区域进行测序，并分析转录因子结合位点的存在情况。通过瞬时转染含有人RPE细胞系ARPE19和D407以及SK-Mel-28和HeLa细胞系上游序列嵌套缺失的荧光素酶报告构建体来检测启动子活性。通过DNase I足迹分析鉴定转录起始位点上游340 bp中存在的特异性DNA-蛋白质结合位点。
序列分析将多态性标记D1S2803定位在RPE65上游区域，并鉴定出一些与编码细胞视黄醛结合蛋白的基因同源的序列。功能分析表明，基础启动子活性由-83至+39的序列赋予，并且在所有测试的细胞系中大致相同，在上游序列的3.6 kb中未检测到其他调控元件。在DNase I足迹分析中鉴定出至少八个受保护区域，包括与预测的TATA盒、AP-4和核因子-1 DNA-蛋白质结合位点相对应的序列。
这些发现确定了RPE65的基础启动子活性，鉴定了作为基因表达正调控因子的潜在顺式作用元件，并表明可能还有其他调控元件参与将基因表达限制在视网膜色素上皮细胞。鉴定上游序列中的启动子元件和遗传标记将有助于筛查视网膜变性患者中可能影响RPE65表达的突变。
