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Transcriptional regulation of the human reduced folate carrier A1/A2 promoter: Identification of critical roles for the USF and GATA families of transcription factors

机译:人类减少的叶酸载体A1 / A2启动子的转录调控:确定USF和GATA转录因子家族的关键作用

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The human reduced folate carrier (hRFC) gene has a complex regulation involving 6 alternatively spliced non-coding exons and promoters (A1/A2, A, B, C, D, and E). The hRFC-A1/A2 promoter is unique in that it transcribes a novel transcript with an in-frame AUG in non-coding exon A1/A2 that encodes a modified hRFC protein with altered transport function. In this report, we characterize the hRFC-A1/A2 promoter in HepG2 human hepatoma cells. By transfecting HepG2 cells with 5' and 3' deletion constructs, a transcriptionally important 270 bp region was identified. Gel shift assays identified transcription factor binding to three E-box elements and one GATA site within this region. These elements were verified by transfections of mutant constructs into HepG2 cells. Cotransfections in Drosophila Mel-2 cells confirmed promoter activation by USF1 and GATA1. A physical association between USF1 and GATA1 was demonstrated by their co-immunoprecipitation. By real time PCR analysis of transfected HepG2 cells, USF1 and GATA1 increased endogenous hRFC-A1/A2 transcripts. Altogether, our results demonstrate a transcriptionally important region in the hRFC-A1/A2 promoter including E-box and GATA elements, and a transactivation by USF1 and GATA1 proteins. Our results further establish the complexity of hRFC regulation, as a means of ensuring adequate folate cofactor transport for cell proliferation. (c) 2005 Elsevier B.V. All rights reserved.
机译:人类减少的叶酸载体(hRFC)基因具有复杂的调控,涉及6个选择性剪​​接的非编码外显子和启动子(A1 / A2,A,B,C,D和E)。 hRFC-A1 / A2启动子的独特之处在于,它在非编码外显子A1 / A2中以框内AUG转录新的转录本,该外显子编码具有改变的转运功能的修饰的hRFC蛋白。在本报告中,我们表征了HepG2人肝癌细胞中的hRFC-A1 / A2启动子。通过用5'和3'缺失构建体转染HepG2细胞,鉴定了一个重要的270 bp转录区域。凝胶移位测定法鉴定了转录因子与该区域内的三个E-box元件和一个GATA位点的结合。通过将突变体构建体转染到HepG2细胞中来验证这些元素。果蝇Mel-2细胞中的共转染证实USF1和GATA1启动子激活。通过USF1和GATA1的共免疫沉淀证明了它们之间的物理联系。通过对转染的HepG2细胞进行实时PCR分析,USF1和GATA1增加了内源性hRFC-A1 / A2转录本。总之,我们的结果证明了hRFC-A1 / A2启动子中的一个重要转录区域,包括E-box和GATA元件,以及USF1和GATA1蛋白的反式激活。我们的结果进一步确定了hRFC调控的复杂性,以此作为确保叶酸辅因子转运以促进细胞增殖的手段。 (c)2005 Elsevier B.V.保留所有权利。

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