Chen Shiming, Wang Qing-Liang, Xu Siqun, Liu Ivy, Li Lili Y, Wang Yufang, Zack Donald J
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
Hum Mol Genet. 2002 Apr 15;11(8):873-84. doi: 10.1093/hmg/11.8.873.
Mutations in the photoreceptor transcription factor cone-rod homeobox (CRX) have been identified in patients with several forms of retinal degenerative disease. To investigate the mechanisms by which these mutations cause photoreceptor degeneration, CRX constructs representing eleven known mutations, as well as a set of C-terminal deletions, were generated and tested for their ability to activate a rhodopsin-luciferase reporter in a transient cell transfection assay. To further define functional domains, several Gal4dbd-Crx fusions were similarly tested using a Gal4 response element containing heterologous promoter. This analysis demonstrated that the C-terminal region, between amino acids 200 and 284, is essential for CRX-mediated transcriptional activation. Consistent with this, four mutants carrying C-terminal truncations demonstrated significantly reduced transcriptional activation. Confirming the importance of the homeodomain (HD), four of the five mutants carrying HD missense mutations displayed altered transactivating activity, either decreased (three) or increased (one). In vitro protein-DNA binding assays (EMSAs) with CRX-HD peptides representing the three HD mutants with decreased transactivating activity, indicated that the alteration was due to reduced, but not abolished, DNA binding to CRX targets. Taken together, these results support the hypothesis that CRX mutations involved in human photoreceptor degeneration act by impairing CRX-mediated transcriptional regulation of the photoreceptor genes. However, a clear relationship between the magnitude of biochemical abnormality and degree of disease severity was not observed, suggesting that other genetic and environmental modifiers may also contribute to the disease phenotype.
在患有多种形式视网膜退行性疾病的患者中，已鉴定出光感受器转录因子视锥 - 视杆同源框（CRX）的突变。为了研究这些突变导致光感受器退化的机制，构建了代表11种已知突变的CRX构建体以及一组C末端缺失构建体，并在瞬时细胞转染试验中测试它们激活视紫红质 - 荧光素酶报告基因的能力。为了进一步确定功能域，使用含有异源启动子的Gal4反应元件对几种Gal4dbd - Crx融合体进行了类似测试。该分析表明，氨基酸200至284之间的C末端区域对于CRX介导的转录激活至关重要。与此一致的是，四个携带C末端截短的突变体显示转录激活显著降低。证实同源结构域（HD）的重要性，五个携带HD错义突变的突变体中有四个表现出改变的反式激活活性，要么降低（三个）要么增加（一个）。用代表三个反式激活活性降低的HD突变体的CRX - HD肽进行的体外蛋白质 - DNA结合试验（EMSA）表明，这种改变是由于与CRX靶标的DNA结合减少但未消除所致。综上所述，这些结果支持这样的假设，即参与人类光感受器退化的CRX突变通过损害CRX介导的光感受器基因转录调控而起作用。然而，未观察到生化异常程度与疾病严重程度之间的明确关系，这表明其他遗传和环境修饰因子也可能导致疾病表型。
