Rowntree R K, Vassaux G, McDowell T L, Howe S, McGuigan A, Phylactides M, Huxley C, Harris A
Paediatric Molecular Genetics, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK.
Hum Mol Genet. 2001 Jul 1;10(14):1455-64. doi: 10.1093/hmg/10.14.1455.
The elements controlling the complex developmental and tissue-specific expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene lie outside the basal promoter region and have not been characterized. We previously identified a tissue-specific DNase I hypersensitive site (DHS) in intron 1 (185 + 10 kb) of the CFTR gene. Here we show that removal of the core element abolishes the activity of this DHS in transient transfection assays of reporter/enhancer gene constructs. We then compared expression from a 310 kb yeast artificial chromosome (YAC) that contains the entire CFTR gene with expression from the same YAC from which the DHS element had been deleted. Stable transfection of a human colon carcinoma cell line showed that transcription from the deleted YAC was reduced by approximately 60%. In transgenic mice, deletion of the intron 1 DHS had no effect on expression in the lung, but reduced expression in the intestine by approximately 60%. Thus, the regulatory element associated with the intron 1 DHS is tissue-specific and is required for normal CFTR expression levels in the intestinal epithelium in vivo.
控制囊性纤维化跨膜传导调节因子（CFTR）基因复杂的发育和组织特异性表达的元件位于基础启动子区域之外，尚未得到鉴定。我们之前在CFTR基因内含子1（185 + 10 kb）中鉴定出一个组织特异性的DNA酶I超敏位点（DHS）。在此我们表明，在报告基因/增强子基因构建体的瞬时转染实验中，去除核心元件会消除该DHS的活性。然后我们比较了包含整个CFTR基因的310 kb酵母人工染色体（YAC）的表达与缺失DHS元件的同一YAC的表达。对人结肠癌细胞系进行稳定转染表明，缺失YAC的转录降低了约60%。在转基因小鼠中，缺失内含子1 DHS对肺中的表达没有影响，但使肠道中的表达降低了约60%。因此，与内含子1 DHS相关的调节元件具有组织特异性，是体内肠道上皮中正常CFTR表达水平所必需的。
