Isolation and characterization of the 5 '-flanking region of the human PDXK gene
Isolation and characterization of the 5 '-flanking region of the human PDXK gene
复制标题
DOI：
10.1016/j.gene.2017.07.044
复制
发表时间：
2017
期刊：
影响因子：
3.5
通讯作者：
Huang LongQuan
中科院分区：
文献类型：
--
作者：
Huang ShuoHao;Liu ZhengQing;Ma ZhenQiao;Zhang JianYun;Huang LongQuan
吡哆醛激酶是5′-磷酸吡哆醛生物合成的关键酶。吡哆醛5′-磷酸是维生素B6的催化活性形式，在超过140种不同的酶反应中作为辅因子。目前尚不清楚激酶合成在细胞中是如何调节的，也没有关于基因启动子的报道。本研究在对人PDXK基因5′端侧翼序列进行生物信息学分析的基础上，通过PCR方法克隆了该基因的启动子序列。通过构建一系列含人PDXK启动子区的荧光素酶表达载体，对该启动子的结构和功能进行了研究。转录起始位点位于ATG翻译起始位点上游198 bp处。一个重要的调控区位于转录起始位点上游的− 665/− 433 bp处。该启动子缺乏典型的TATA盒，但含有三个GC盒和一个E盒。缺失和突变实验表明，转录因子Sp1结合位点C（-553/-543）对于维持强大的启动子活性至关重要。通过RNA干扰和染色质免疫沉淀分析进一步证实Sp1参与PDXK基因表达的调控。
Pyridoxal kinase is a key enzyme for the biosynthesis of pyridoxal 5′-phosphate. Pyridoxal 5′-phosphate is the catalytically active form of vitamin B6, and acts as a cofactor in > 140 different enzyme reactions. It is still unknown how the kinase synthesis is regulated in the cells, and nothing has been reported about the gene promoter. In the present study, based on the bioinformatics analysis of the 5′-flanking region of the humanPDXKgene, we cloned the promoter region by PCR. Through the construction of a series of luciferase expression vectors containing the humanPDXKpromoter region, we characterized the promoter in terms of its structure and function. The transcription start site is at 198 bp upstream of the ATG translation initiation site. An important regulatory region is located at − 665/− 433 bp upstream of the transcription start site. The promoter lacks the canonical TATA box, but contains three GC-boxes and one E-box. A deletion and mutation experiment revealed that the transcription factor Sp1 binding site C (− 553/− 543) is critical in maintaining the robust promoter activity. Knockdown ofSp1by RNA interference and chromatin immunoprecipitation analysis further proved that the Sp1 is involved in the regulation of thePDXKgene expression.