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首页> 外文期刊>Biochemistry >STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE HUMAN KB CELL FOLATE RECEPTOR GENE P4 PROMOTER - COOPERATION OF THREE CLUSTERED SP1-BINDING SITES WITH INITIATOR REGION FOR BASAL PROMOTER ACTIVITY
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STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE HUMAN KB CELL FOLATE RECEPTOR GENE P4 PROMOTER - COOPERATION OF THREE CLUSTERED SP1-BINDING SITES WITH INITIATOR REGION FOR BASAL PROMOTER ACTIVITY

机译：人KB细胞叶酸受体基因P4启动子的结构和功能分析-三种带有SP1结合位点的聚簇位点与基础激活子区域的合作。

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The human folate receptors (hFRs) are important in the cellular accumulation of folates and antifolates. We described the structure of the human KB cell FR (hFR-KB) gene and identified two discrete promoter regions (P1 and P4) upstream from exons 1 and 4, respectively (Elwood et al., 1993). To further understand the molecular basis of hFR expression, we have now analyzed the basal transcription of the P4 promoter localized upstream of a major transcription start site. The sequence upstream from exon 4 contains several potential transcriptional factor-binding sites and a consensus initiator region sequence at the transcription start site but does not contain canonical TATA or CAAT boxes. While deletion of a 5' flanking sequence from nt - 1023 to nt - 605 of P4 promoter region decreases the luciferase reporter gene expression in KB cells to 54 - 70% of control construct, the removal of the sequence between nt - 292 and nt - 46 markedly decreases the activity to 3%. DNase I footprints and competitive mobility shift and supershift mobility assays indicate that Spl or Spl-related nuclear protein(s) bind to three clustered GC-rich regions within the sequence between nt - 292 and nt - 46 of the hFR-KB P4 promoter. Both in vitro and in vivo analyses of the expression of promoter constructs containing site-specific mutation(s) of these three Spl-binding sites and initiator sequence demonstrate that each of three Spl sites and the initiator sequence are required for optimum promoter activity and that they interact cooperatively in this P4 promoter of the hFR-KB gene.
机译：人叶酸受体（hFRs）在叶酸和抗叶酸的细胞积累中很重要。我们描述了人类KB细胞FR（hFR-KB）基因的结构，并确定了分别位于外显子1和4上游的两个离散的启动子区域（P1和P4）（Elwood等，1993）。为了进一步了解hFR表达的分子基础，我们现在分析了位于主要转录起始位点上游的P4启动子的基础转录。外显子4上游的序列在转录起始位点包含几个潜在的转录因子结合位点和共有启动子区域序列，但不包含规范的TATA或CAAT框。虽然从P4启动子区域的nt-1023到nt-605的5'侧翼序列的缺失使KB细胞中萤光素酶报告基因的表达降低至对照构建体的54-70％，但nt-292和nt-之间的序列被去除。 46明显降低了活性至3％。 DNase I足迹以及竞争性迁移和超迁移迁移试验表明，Spl或Spl相关核蛋白与hFR-KB P4启动子的nt-292和nt-46之间的序列中的三个簇状GC富集区域结合。对包含这三个Spl结合位点和启动子序列的位点特异性突变的启动子构建体表达的体外和体内分析均表明，三个Spl位点和启动子序列中的每一个对于最佳启动子活性都是必需的，并且它们在hFR-KB基因的P4启动子中协同作用。

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