Bioinformatic and functional analysis of promoter region of human SLC25A13 gene
Bioinformatic and functional analysis of promoter region of human SLC25A13 gene
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人SLC25A13基因启动子区的生物信息及功能分析
DOI：
10.1016/j.gene.2019.01.023
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发表时间：
2019-04-20
期刊：
影响因子：
3.5
通讯作者：
Zhou, Qing-Hua
中科院分区：
文献类型：
--
作者：
Chen, Jun-Lin;Zhang, Zhan-Hui;Zhou, Qing-Hua
人SLC25A13基因编码肝型天冬氨酸/谷氨酸载体亚型2（AGC2，俗称citrin），在人肝细胞的主要NADH穿梭中发挥关键作用。双等位基因 SLC25A13 突变导致 Citrin 缺乏 (CD)。为了鉴定SLC25A13基因的重要调控区并阐明潜在的启动子突变如何影响citrin表达，我们进行了启动子缺失分析，并建立了携带荧光素酶基因的SLC25A13启动子的报告构建体，该启动子包含位于假定的转录因子结合位点的多个突变。使用双荧光素酶报告基因检测系统测量所有启动子构建体的荧光素酶活性。生物信息分析表明，SLC25A13基因启动子缺少TATA盒和明显典型的起始元件，但含有一个CCAAT盒和两个GC盒。启动子缺失分析证实，上游 ATG -221 至 -1 区域对于 SLC25A13 维持启动子活性至关重要。我们利用双荧光素酶报告系统作为功能分析模型，初步评估了人工构建的启动子突变对citrin表达的影响，我们的分析表明，突变的假定CCAAT盒和GC盒可以显着影响citrin表达。我们的研究证实了影响HL7702细胞中citrin表达的重要SLC25A13启动子区域，并构建了功能分析模型。这项工作可能有助于进一步鉴定启动子区域导致 CD 的致病突变。
The human SLC25A13 gene encodes the liver type aspartate/glutamate carrier isoform 2 (AGC2, commonly named as citrin), which plays a key role in the main NADH-shuttle of human hepatocyte. Biallelic SLC25A13 mutations result in Citrin deficiency (CD). In order to identify the important regulatory region of SLC25A13 gene and elucidate the way how potential promoter mutations affect the citrin expression, we performed promoter deletion analysis and established the reporter constructs of luciferase gene-carrying SLC25A13 promoter containing several mutations located in putative transcription factor-binding sites. The luciferase activities of all promoter constructs were measured using a Dual-Luciferase Reporter Assay System. Bioinformatic analysis showed that the promoter of SLC25A13 gene lacks TATA box and obviously typical initiator element, but contains a CCAAT box and two GC box. Promoter deletion analysis confirmed the region from -221 to -1 upstream ATG was essential for SLC25A13 to maintain the promoter activity. We utilized dual-luciferase reporter system as function analytical model to tentatively assess the effect of artificially constructed promoter mutations on citrin expression, and our analysis revealed that mutated putative CCAAT box and GC box could significantly affect the citrin expression. Our study confirmed the important SLC25A13 promoter regions that influenced citrin expression in HL7702 cells, and constructed a function analytical model. This work may be useful to further identify the pathogenic mutations leading to CD in the promoter region.