Elements regulating the transcription of human interstitial retinoid-binding protein (IRBP) gene in cultured retinoblastoma cells
Elements regulating the transcription of human interstitial retinoid-binding protein (IRBP) gene in cultured retinoblastoma cells
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DOI：
10.1076/ceyr.18.4.283.5360
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发表时间：
1999-04-01
影响因子：
2
通讯作者：
Fong, WB
中科院分区：
文献类型：
--
作者：
Fong, SL;Fong, WB
目的.鉴定人IRBP基因表达的顺式作用元件和反式作用因子。采用瞬时转染WERI-Rb 1和HeLa细胞、DNA酶1足迹法、凝胶迁移率改变法和酵母单杂交系统对人IRBP基因表达调控元件进行了研究。-1620和-1411之间的区域显示具有增强子特性。使用来自WERI-Rb 1和HeLa细胞的核提取物，在近端启动子区（-206至+68）中鉴定出四个足迹。在酵母单杂交系统中，核心启动子元件IP 1与OTX 2结合。OTX 2通过共转染HeLa细胞，可以反式激活irbp启动子。IP 2（从-119到-86）和IP 3（从-183到-147）的功能仍有待确定。HeLa细胞特异性足迹IP 4（-202 ~-180）区域可沉默OTX 2对irbp启动子的反式激活。irbp的5 '侧翼区含有增强子序列。核心启动子上游可能存在沉默子，可能抑制irbp在HeLa细胞中的表达。当使用近端启动子通过酵母单杂交系统鉴定人视网膜文库中的结合蛋白时，9个鉴定的克隆含有同源结构域蛋白OTX 2的cDNA序列。由于没有发现同源结构域蛋白CRX的克隆，并且由于OTX 2可以在正常非表达的HeLa细胞中转录激活irbp，因此OTX 2而不是CRX可能是人类光感受器中irbp的转录激活因子。
Purpose. To identify cis-acting elements and trans-acting factors involved in the expression of human IRBP gene.Methods. Transient transfection of WERI-Rb1 and HeLa cells, DNase 1 footprinting, gel mobility-shift assay and yeast one-hybrid system were used to study the regulatory elements that are involved in the expression of human IRBP gene.Results. A region between -1620 and -1411 was shown to have enhancer properties. Using nuclear extracts from WERI-Rb1 and HeLa cells, four footprints were identified in the proximal promoter region (-206 to +68). The core promoter element IP1 binds to OTX2 in the yeast one-hybrid system. By cotransfecting HeLa cells, OTX2 could transactivate the irbp promoter. The functions of IP2 (from -119 to -86) and IP3 (from -183 to -147) remain to be determined. The region containing the HeLa cell-specific footprint IP4 (from -202 to -180) could silence the OTX2 transactivation of the irbp promoter.Conclusion. The 5'-flanking region of irbp contains an enhancer sequence. The possible silencer upstream from the core promoter may serve to suppress expression of irbp in HeLa cells. When the proximal promoter is used to identify binding proteins in a human retina library by the yeast one hybrid system, nine of the identified clones contained the cDNA sequence for the homeodomain protein OTX2. Since no clones for the homeodomain protein CRX were found, and since OTX2 can transcriptionally activate irbp in normally non-expressing HeLa cells, it is possible that OTX2 rather than CRX is the transcriptional activator for irbp in human photoreceptors.