Human Molecular Genetics Program, Children's Memorial Research Center, Chicago, IL 60614, USA.
Biochem J. 2012 Sep 1;446(2):203-12. doi: 10.1042/BJ20120693.
A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene.
CFTR（囊性纤维化跨膜电导调节因子）基因的一个关键顺式调控元件位于 11 号内含子中，距离启动子 100kb 远，与启动子相互作用。该序列包含一个肠道选择性增强子，并与增强子特征蛋白（如 p300）以及组织特异性 TF（转录因子）相关联。在本研究中，我们确定了招募到这个元件的关键 TF，并证明了它们在调节 CFTR 表达中的重要性。体外 DNase I 足迹法和电泳迁移率变动分析（EMSA）鉴定出四个细胞类型选择性区域，这些区域在体外与 TF 结合。ChIP（染色质免疫沉淀）鉴定出 FOXA1/A2（叉头框 A1/A2）、HNF1（肝细胞核因子 1）和 CDX2（尾型同源盒 2）是体内相互作用的转录因子。当通过报告基因检测测量时，其内含子 11 核心结合位点的突变会损害其增强子活性。此外，siRNA（小干扰 RNA）介导的 CDX2 敲低会导致肠细胞内源性 CFTR 转录显著减少，表明该因子对于维持这些细胞中 CFTR 高水平表达至关重要。ChIP 数据还表明，这些 TF 与 CFTR 基因座上的多个顺式调控元件相互作用，暗示它们在肠道基因表达中具有更广泛的作用。
