Park Hae Jeong, Lee Soojung, Ju Eunji, Jones Jayre A, Choi Inyeong
Department of Pharmacology, Kyung Hee University School of Medicine, Seoul, South Korea; and.
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia.
Physiol Genomics. 2017 Mar 1;49(3):167-176. doi: 10.1152/physiolgenomics.00112.2016. Epub 2017 Jan 13.
Genome-wide association studies have identified the single nucleotide polymorphism (SNP) rs3278 in the human gene as one of the marker loci for addiction vulnerability. This marker is located in an intron of the gene, and its genomic role has been unknown. In this study, we examined rs3278 and three adjacent SNPs prevalent in alcoholics for their effects on an alternative promoter that would lead to the production of the NH-terminally truncated protein NBCn1ΔN450, missing the first 450 amino acids. Analysis of the transcription start site database and a promoter prediction algorithm identified a cluster of three promoters in intron 7 and two short CpG-rich sites in intron 6. The promoter closest to rs3278 showed strong transcription activity in luciferase reporter gene assays. Major-to-minor allele substitution at rs3278 resulted in increased transcription activity. Equivalent substitutions at adjacent rs3772723 (intron 7) and rs13077400 (exon 8) had negligible effect; however, the substitution at nonsynonymous rs3755652 (exon 8) increased the activity by more than twofold. The concomitant substitution at rs3278/rs3755652 produced an additive effect. The rs3755652 had more profound effects on the promoter than the upstream regulatory CpG sites. The amino acid change E326K caused by rs3755652 had negligible effect on transporter function. In HEK 293 cells, NBCn1ΔN450 was expressed in plasma membranes, but at significantly lower levels than the nontruncated NBCn1-E. The pH change mediated by NBCn1ΔN450 was also low. We conclude that rs3278 and rs3755652 stimulate an alternative transcription of the gene, increasing the production of a defective transporter.
全基因组关联研究已确定人类基因中的单核苷酸多态性（SNP）rs3278是成瘾易感性的标记位点之一。该标记位于基因的一个内含子中，其基因组作用尚不清楚。在本研究中，我们检测了rs3278以及在酗酒者中普遍存在的三个相邻SNP对一个替代启动子的影响，该启动子会导致产生N端截短的蛋白NBCn1ΔN450，缺失前450个氨基酸。对转录起始位点数据库和启动子预测算法的分析确定了内含子7中的三个启动子簇以及内含子6中的两个富含CpG的短位点。最接近rs3278的启动子在荧光素酶报告基因检测中显示出较强的转录活性。rs3278处的主要等位基因到次要等位基因的替换导致转录活性增加。相邻的rs3772723（内含子7）和rs13077400（外显子8）处的等效替换影响可忽略不计；然而，非同义rs3755652（外显子8）处的替换使活性增加了两倍多。rs3278/rs3755652处的同时替换产生了累加效应。rs3755652对启动子的影响比上游调控的CpG位点更深远。rs3755652引起的氨基酸变化E326K对转运蛋白功能的影响可忽略不计。在HEK 293细胞中，NBCn1ΔN450在质膜中表达，但水平明显低于未截短的NBCn1-E。由NBCn1ΔN450介导的pH变化也很低。我们得出结论，rs3278和rs3755652刺激了该基因的替代转录，增加了有缺陷转运蛋白的产生。
