Functional characterization of a-100_-102delAAG deletion-insertion polymorphism in the promoter region of the HTR3B gene
Functional characterization of a-100_-102delAAG deletion-insertion polymorphism in the promoter region of the HTR3B gene
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DOI：
10.1097/fpc.0b013e3282f51092
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发表时间：
2008-03-01
影响因子：
2.6
通讯作者：
Brockmoeller, Juergen
中科院分区：
文献类型：
--
作者：
Meineke, Cornelia;Tzvetkov, Mladen Vassilev;Brockmoeller, Juergen
目的HTR 3B基因编码3型5-羟色胺受体（5-HT 3）的B亚单位。启动子区的-100--102delAAG缺失与止吐药物反应不良和双相情感障碍易感性相关。这些协会的分子机制，然而，仍然不清楚。方法我们进行了电泳迁移率变化和荧光素酶报告基因检测，以阐明这种多态性对PC-12和HEK 293细胞中HTR 3B启动子活性的影响。结果缺失图谱分析表明，HTR 3B启动子-100 ~-102delAAG多态性位点附近的序列具有明显的启动子活性。电泳迁移率变动分析表明，差异结合的核蛋白的多态性DNA区域具有较强的结合插入比缺失等位基因。与相应的插入构建体相比，携带缺失等位基因的天然启动子的活性在PC-12中高25%（P=0.016），在HEK细胞中高40%（P=0.016）。一式三份串联携带缺失等位基因的构建体显示出比携带插入等位基因的构建体高43%（PC-12细胞，P=0.002）和28%（HEK 293细胞，P=0.015）的活性。该多态性与HTR 3A和HTR 3B中已知的氨基酸替换无关。结论HTR 3B启动子-100 ~-1102delAAG 3bp缺失在体外可提高HTR 3B启动子活性。这对所产生的5-HT 3受体的结构和功能的影响仍有待阐明。
Objective The HTR3B gene encodes the B-subunit of the type 3 serotonin receptor (5-HT3). A -100_-102delAAG deletion in the promoter region has been associated with poor response to antiemetic medication and susceptibility to bipolar affective disorders. The molecular mechanisms underlying these associations, however, remained unclear.Methods We performed electrophoretic mobility shift and luciferase reporter gene assays to elucidate the effect of this polymorphism on the HTR3B promoter activity in PC-12 and HEK293 cells. The reporter constructs carried a 2171 bp fragment of the native HTR3B promoter or 30 bp of the polymorphic locus in tandem triplication upstream of the thymidine kinase minimal promoter.Results Deletion mapping indicated that the sequence around the -100_-102delAAG polymorphism had significant promoter activity. Electrophoretic mobility shift assays indicated differential binding of nuclear proteins to the polymorphic DNA region with stronger binding to the insertion than to the deletion allele. The activity of the native promoter carrying the deletion allele was 25% higher in PC-12 (P=0.016) and 40% higher in HEK cells (P=0.016) compared with the respective insertion construct. Constructs carrying the deletion allele in tandem triplicates showed 43% (PC-12 cells, P=0.002) and 28% (HEK293 cells, P=0.015) higher activity than those carrying the insertion allele. The polymorphism was not linked with known amino acid substitutions in HTR3A and HTR3B.Conclusions The -100_-1102delAAG 3bp deletion increases the HTR3B promoter activity in vitro. The consequences of this for the structure and the function of the resulting 5-HT3 receptors remain to be elucidated.