Transcriptional networks driving enhancer function in the CFTR gene
Transcriptional networks driving enhancer function in the CFTR gene
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DOI：
10.1042/bj20120693
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发表时间：
2012-09-01
影响因子：
4.1
通讯作者：
Harris, Ann
中科院分区：
文献类型：
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作者：
Kerschner, Jenny L.;Harris, Ann
囊性纤维化跨膜传导调节因子（CFTR）基因的一个关键顺式调控元件位于内含子11，100 kb的启动子远端，它与之相互作用。该序列包含一个丝氨酸选择性增强子，并与增强子标签蛋白，如p300，以及组织特异性TF（转录因子）。在本研究中，我们确定了关键的转录因子，招募到这个元素，并证明其在调节CFTR表达的重要性。体外DNA酶I足迹法和EMSA（电泳迁移率变化测定）确定了四个细胞类型选择性区域，结合TF在体外。ChIP（染色质免疫沉淀）鉴定FOXA 1/A2（叉头框A1/A2）、HNF 1（肝细胞核因子1）和CDX 2（尾型同源框2）为体内反式相互作用因子。当通过报告基因测定测量时，内含子11核心中的它们的结合位点的突变损害了其增强子活性。此外，siRNA（小干扰RNA）介导的CDX 2敲低导致肠细胞中内源性CFTR转录的显著减少，表明该因子对于维持这些细胞中高水平的CFTR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，暗示了该基因在肠道表达中的更全面的作用。
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.