Twenty-one-base-pair insertion polymorphism creates an enhancer element and potentiates SLC6A1 GABA transporter promoter activity.
Twenty-one-base-pair insertion polymorphism creates an enhancer element and potentiates SLC6A1 GABA transporter promoter activity.
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DOI：
10.1097/fpc.0b013e328318b21a
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发表时间：
2009-01
影响因子：
2.6
通讯作者：
Simen AA
中科院分区：
文献类型：
--
作者：
Hirunsatit R;George ED;Lipska BK;Elwafi HM;Sander L;Yrigollen CM;Gelernter J;Grigorenko EL;Lappalainen J;Mane S;Nairn AC;Kleinman JE;Simen AA
钠依赖性和氯依赖性γ-氨基丁酸（GABA）转运蛋白1（SLC 6A 1）是许多具有临床重要性的药物的靶点，也是突触GABA浓度的主要决定因素。我们重新测序的人SLC 6A 1基因之前，发现了一个新的21 bp插入在预测的启动子区域，创建第二个串联拷贝的序列。在这里，我们试图确定这种变化的功能相关性。我们使用了报告分析，迁移率变动分析，定量PCR和蛋白质组学方法，以及死后表达分析这项工作。报告基因分析表明，插入等位基因显着增加启动子活性在多个细胞系。锌指转录因子ZNF 148被发现显着的反式激活启动子和增加表达时，过表达，但不能解释的两个等位基因的启动子之间的活性差异。插入序列的拷贝数与下游启动子的指数增加的活性相关，表明插入序列在以多个拷贝存在时具有增强子活性。发现SLC 6A 1启动子基因型预测人死后海马样品中SLC 6A 1 RNA表达。这些结果表明，插入多态性导致SLC 6A 1启动子活性增加，部分原因是当作为多个拷贝存在时产生增强子元件。在这项研究中，来自坦桑尼亚的基因分型个体表明插入等位基因起源于非洲。考虑到插入对启动子活性的影响，这种相对常见的多态性可证明可用于预测非洲血统个体对SLC 6A 1药理学调节剂的临床反应以及GABA能功能。
Sodium-dependent and chloride-dependent γ-aminobutyric acid (GABA) transporter 1 (SLC6A1) is the target of a number of drugs of clinical importance and is a major determinant of synaptic GABA concentrations. We resequenced the human SLC6A1 gene previously and discovered a novel 21 bp insertion in the predicted promoter region that creates a second tandem copy of the sequence. Here we sought to determine the functional relevance of this variation. We used reporter assays, mobility shift assays, quantitative PCR, and proteomics methods as well as postmortem expression analysis for this work. Reporter assays showed that the insertion allele significantly increases promoter activity in multiple cell lines. The zinc finger transcription factor ZNF148 was found to significantly transactivate the promoter and increase expression when overexpressed but could not account for the differences in activity between the two alleles of the promoter. Copy number of the insertion sequence was associated with exponentially increasing activity of a downstream promoter, suggesting that the insertion sequence has enhancer activity when present in multiple copies. SLC6A1 promoter genotype was found to predict SLC6A1 RNA expression in human postmortem hippocampal samples. These results suggest that the insertion polymorphism leads to increased SLC6A1 promoter activity because, in part, of creation of an enhancer element when present as multiple copies. Genotyping individuals from Tanzania in this study suggested that the insertion allele has its origin in Africa. On account of the effect of the insertion on promoter activity, this relatively common polymorphism may prove useful in predicting clinical response to pharmacological modulators of SLC6A1 as well as GABAergic function in individuals of African descent.