人SATB1基因5′上游序列的转录激活功能分析

在对人SATB1基因进行生物信息学分析的基础上 ,采用PCR技术 ,扩增人基因组DNA中SATB1基因 5′上游序列的 - 2 95 5～ - 9片段 ,构建了 3个分别由SATB1基因 5′上游 - 2 95 5～ - 9,- 172 7～ - 9和 - 76 0～ - 9序列片段驱动的报告载体 -pGL3 SP2 94 6 luc ,pGL3 SP1718 luc和pGL3 SP75 1 luc ,分别瞬时转染JurkatT ,K5 6 2 ,U937和HeLa细胞 ,通过测定荧光素酶的表达活性 ,观察SATB1基因 5′上游序列片段 3个删除突变体在不同细胞内活性的差异 .结果显示 ,SATB1上游序列- 2 95 5 - 9在 4种细胞中的转录激活能力为U937>JurkatT >K5 6 2 ,在HeLa细胞中基本无激活 ,提示SATB1的转录激活可能具有一定的细胞类型特异性 .3种 5′删除突变体转录激活性由大至小顺序为 - 76 0 - 9>- 2 95 5 - 9>- 172 7 - 9,提示SATB1的核心启动子可能存在于其 5′上游序列的- 76 0至 - 9bp区域中 .

Identification of Core Promoter Sequences in Upstream Promoter Sequeces Responsible for Transcriptional Regulation of Human SATB1 Gene in vitro

The biological information of human special A-T rich sequence binding protein 1(SATB1) was analyzed.SATB1,a DNA-binding protein expressed predominantly in thymocytes,recognizes an ATC sequence context of promoter sequence of target genes.The current study employed three luciferase reporters containing truncated different promoter sequences in order to identify the minimal promoter sequence of SATB1 gene.Promoter constructs spanned -2955～-9,-1727～-9 and -760～-9 of 5′ upstream sequences from human SATB1 gene. Then pGL3-SP2946-luc,pGL3-SP1718-luc and pGL3-SP751-luc were transfected into Jurkat T,K562,U937 and HeLa cells transiently and their activity were observed, respectively. Results showed that the activity of -2955～-9 of 5′ upstream sequences from human SATB1 gene in 4 different type of cells is U937>Jurkat T>K562 and showed no obvious activity in HeLa cells, and demonstrated that SATB1 promoter might drive gene expression with cell-type specificity. Three different deleted mutants' transfected activity in 4 cell-lines is -760/-9>-2955/-9>-1727/-9,implied that its core promoter region might located in -760～-9 bp of 5′ upstream sequences of human SATB1 gene.