Identification of two promoters for human d-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2.
Identification of two promoters for human d-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2.
复制标题
人 d-氨基酸氧化酶基因两个启动子的鉴定：PAX5/PAX2 介导的差异启动子调控的意义。
DOI：
10.1093/jb/mvu084
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Fukui K.
中科院分区：
文献类型：
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作者：
Tran DH;Shishido Y;Chung SP;Trinh HT;Yorita K;Sakai T;Fukui K.
d-氨基酸氧化酶（DAO）是一种代谢d-氨基酸的黄素酶。到目前为止，DAO的表达机制仍不清楚。我们使用荧光素酶报告系统对人类DAO（hDAO）启动子活性的评估表明外显子1的近端上游区域（-237/+1）具有启动子活性（P1）。有趣的是，我们在外显子2的近端上游区域（+4，126/+4，929）（P2）中鉴定了替代启动子。该替代启动子具有比P1更强的活性。我们的研究结果还揭示了内含子1中的负调控片段（+1，163/+1，940）;这将与P1和P2协同作用。生物信息学分析阐明了物种间转录因子PAX 5家族结合位点的保守性。这些位点（−60/−31）和（+4，464/+4，493）分别位于hDAO的P1和P2。凝胶位移分析表明，P1含有PAX 5结合位点（-60/-31），而P2有三个配对盒基因2（PAX 2）和配对盒基因5（PAX 5）结合位点。基于定点突变实验，推测PAX 5家族通过调节P1启动子活性和激活P2启动子活性来调节hDAO转录的双重作用。总之，我们的数据表明，差异调节hDAO表达的两个启动子，其活动可能是由PAX 2和PAX 5的结合调制。
d-Amino acid oxidase (DAO) is a flavoenzyme that metabolizesd-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (−237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (−60/−31) and (+4,464/+4,493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (−60/−31) for PAX5 binding while P2 has three sites for both paired box gene 2 (PAX2) and paired box gene 5 (PAX5) binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5.