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首页> 外文期刊>Molecular reproduction and development >Involving AP-2 transcription factor in connexin 26 up-regulation during pregnancy and lactation.
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Involving AP-2 transcription factor in connexin 26 up-regulation during pregnancy and lactation.

机译:在怀孕和哺乳期间,AP-2转录因子参与连接蛋白26的上调。

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摘要

Gap junction connexin 26 (Cx26) is up-regulated in mammary epithelial cells during pregnancy and lactation. To understand the transcriptional regulation of Cx26, we identified a protected DNase I footprint region (-140 to -113) in the rat Cx26 promoter. This rCx26 Promoter Footprinting Region, or CPFR, contains an Sp binding site (CCGCCC) overlapping with an AP-2 binding site (GCCCGCGGC), and is evolutionarily conserved. Nuclear extracts from rat mammary glands and human MCF-10 mammary epithelial cells formed protein-DNA complexes with the labeled CPFR probe in the electrophoretic mobility shift assay (EMSA), and these complexes were markedly enhanced during pregnancy and lactation. Antibody supershift analysis further identified the presence of Sp1, Sp3, and AP-2 in these binding complexes. Human mammary epithelial MCF-10A and MCF-12A cells were transiently transfected with chimeric mutant rCx26 promoter/luciferase reporter constructs, and luciferase activities measured. Mutations along the CPFR fragment drastically reduced the promoter activity, specially at the Sp/AP-2 overlapping site. Cotransfection of AP-2 with rCx26 promoter/reporter constructs into MCF-10 cells markedly induced the reporter activity. These data infer that AP-2, along with previously reported Sp transcription factors, is involved in the up-regulation of Cx26 gene during pregnancy and lactation.
机译:在妊娠和哺乳期间,乳腺上皮细胞中的间隙连接连接蛋白26(Cx26)上调。为了解Cx26的转录调控,我们在大鼠Cx26启动子中鉴定了一个受保护的DNase I足迹区域(-140至-113)。该rCx26启动子足迹区域或CPFR包含一个Sp结合位点(CCGCCC)和一个AP-2结合位点(GCCCGCGGC),并且在进化上是保守的。大鼠乳腺和人MCF-10乳腺上皮细胞的核提取物在电泳迁移率变动分析(EMSA)中与标记的CPFR探针形成蛋白质-DNA复合物,并且这些复合物在怀孕和哺乳期显着增强。抗体超位移分析进一步确定了这些结合复合物中Sp1,Sp3和AP-2的存在。用嵌合突变体rCx26启动子/荧光素酶报告基因构建体瞬时转染人乳腺上皮MCF-10A和MCF-12A细胞,并测量荧光素酶活性。沿着CPFR片段的突变会大大降低启动子活性,特别是在Sp / AP-2重叠位点。将AP-2与rCx26启动子/报告子构建体共转染到MCF-10细胞中可明显诱导报告子活性。这些数据表明,AP-2与先前报道的Sp转录因子一起在妊娠和哺乳期间参与了Cx26基因的上调。

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