Kraus Dominik, Karlstetter Marcus, Walczak Yana, Hilfinger Daniela, Langmann Thomas, Weber Bernhard H F
Institute of Human Genetics, University of Regensburg, Germany.
Biochim Biophys Acta. 2011 Apr-Jun;1809(4-6):245-54. doi: 10.1016/j.bbagrm.2011.03.001. Epub 2011 Mar 23.
X-linked juvenile retinoschisis (XLRS) is an orphan retinal disease in males caused by mutations in the RS1 gene. Previously we have characterized cone-rod homeobox (CRX)-responsive elements in the promoter region of RS1 driving selective gene expression in the retina. Here, we expanded our identification and functional analysis of cis-regulatory elements controlling quantitative expression of RS1 in vitro and in vivo. Sequence analysis identified a CpG island 3kb upstream of the transcription start site (TSS). In addition, chromatin immunoprecipitation coupled to microarrays (ChIP-Chip) targeting the retinal transcription factor CRX was performed. Thereby, we identified a second CRX-bound region (CBR2) in the first intron of RS1 which contains six evolutionarily conserved CRX binding motifs. In vitro luciferase reporter gene assays and dsRed reporter electroporation of mouse retinal organ cultures demonstrated a strong constitutive and orientation-independent enhancing effect of the upstream CpG island. The intronic CBR2 potently suppressed CBR1-driven RS1 promoter activity in vitro but failed to regulate a CBR1-reporter in short-term cultured mouse retinae. We conclude that a CpG island enhancer and two CBRs may act in a combinatorial fashion to fine-tune RS1 transcript levels in the retina.
X连锁青少年视网膜劈裂症（XLRS）是一种男性罕见的视网膜疾病，由RS1基因突变引起。此前我们已经对RS1基因启动子区域中驱动视网膜选择性基因表达的视锥-视杆同源框（CRX）反应元件进行了表征。在此，我们扩展了对体外和体内控制RS1定量表达的顺式调控元件的鉴定和功能分析。序列分析在转录起始位点（TSS）上游3kb处鉴定出一个CpG岛。此外，还进行了针对视网膜转录因子CRX的染色质免疫沉淀与微阵列分析（ChIP-Chip）。由此，我们在RS1的第一个内含子中鉴定出第二个CRX结合区域（CBR2），其包含六个进化上保守的CRX结合基序。体外荧光素酶报告基因检测以及小鼠视网膜器官培养物的dsRed报告基因电穿孔实验表明，上游CpG岛具有强大的组成型且不依赖方向的增强作用。内含子CBR2在体外能有效抑制CBR1驱动的RS1启动子活性，但在短期培养的小鼠视网膜中未能调节CBR1报告基因。我们得出结论，一个CpG岛增强子和两个CBR可能以组合方式发挥作用，以微调视网膜中RS1的转录水平。
