大鼠γ谷氨酰半胱氨酸合成酶催化亚单位基因5′端调控序列初步分析

Preliminary analysis of the 5′-flanking region of rat γ-glutamylcysteine synthetase catalytic subunit gene

目的 分析大鼠γ谷氨酰半胱氨酸合成酶(γ-GCS)催化亚单位 (GCLC)基因 5′端调控区域和相应转录因子的特性。方法 克隆 1 760bp大鼠GCLC上游调控基因并构建含荧光素酶基因的报道载体GCLC-Luc(GCLC/pGL-3),利用外切核酸酶Ⅲ将大鼠GCLC基因的 5′端序列单向切成不同长度的缺失体,将所构建的GCLC-Luc及其 11个缺失体转染大鼠肺泡上皮细胞,通过测量转染后细胞的荧光素酶活性确定该基因的调控区域,并通过转录因子分析软件分析出可能与这些调控区域结合的转录因子,最后通过电泳迁移率改变实验 (EMSA)以明确该调控区的顺式元件和转录因子。结果 实验成功地克隆出大鼠GCLC上游调控基因及其报道载体GCLC-Luc及GCLC-Luc的 11个缺失体。将GCLC-Luc及其缺失体转染肺泡上皮细胞并分析荧光素酶活性:GCLC Luc(-1 758 /+2 Luc),缺失体 1(-1 231 /+2 Luc),缺失体 2(-1 108 /+2 Luc),缺失体 3(-1 087 /+2 Luc),缺失体 4(-876 /+2 Luc),缺失体 5(-745 /+2 Luc),缺失体 6(-705 /+2 Luc),缺失体 8(-613 /+2 Luc),缺失体 9(-595 /+2 Luc),缺失体 10( -403 /+2 Luc)和缺失体 11( -111 /+2 Luc)的荧光素酶值分别为(90 012±2 445)、(77 652±840)、(149 927±4 915)、(71 588±1 108)、(99 283±2 612)、(75 443±1 438)。

Objective To analyze the characteristic of regulatory elements and corresponding transcriptional factors in the 5′-flanking region of rat gamma-glutamylcysteine synthetase(γ-GCS) catalytic subunit (GCLC)gene. Methods A 1 760 bp 5′-flanking region of the rat GCLC was cloned and constructed into pGL-3 enhancer vector which includes luciferase reporter gene. Exonuclease Ⅲ was used to cut the 5′-flanking region of rat GCLC gene unidirectionally into deletion mutants of different length,GCLC-Luc and its deletion mutants were used to transfect rat alveolar epithelium cells CCL-149,then the regulatory region of the gene was determined by luciferase activity assay of the transfected cells. Analysis of the transcription-factor-binding site was done using Transcription Factor Search software to indicate possible transcriptional factors. Electrophoresis mobility shift assay(EMSA)was used to determine the cis-acting elements and transcriptional factors in these regulatory regions. Results The experiment cloned the upstream regulatory sequence of rat GCLC gene and its reporter vector GCLC-Luc,as well as 11 deletion mutants of GCLC-Luc. Luciferase activity assay of the cells transfected by GCLC-Luc(-1 758/+2-Luc),mutant 1((-1 231/+2-Luc),)mutant 2(-1 108/+2-Luc),mutant 3(-1 087/+2-Luc),mutant 4((-876/+2-Luc),)mutant 5(-745/+2-Luc),mutant 6(-705/+2-Luc),mutant 8(-613/+2-Luc),mutant 9(-595/+2-Luc),mutant 10(-403/+2-Luc) and mutant 11(-111/+2-Luc) were (90 012±2 445),(77 652±840),(149 927±4 915),(71 588±1 108),(99 283±2 612),(75 443±1 438),(282 772±7 046),(96 891±2 275),(148 917±5 966),(258 991±5 015) and (895±49)U,respectively. EMSA proved that activated protein-1(AP-1) and nuclear factor-κB (NF-κB) can bind to the region of -403 to -111 bp;nuclear factor-1(NF-1) and CCAAT/enhancer binding protein(C/EBP) can bind to the region of -705 to -613 bp;and upstream stimulatory factor(USF) can bind to the region of (-745) to -705 bp. Conclusions Two DNA regions -403 to -111 bp and -705 to -613 bp of GCLC gene,which can be bound by transcriptional factors such as NF-1,C/EBP,AP-1,and NF-κB on EMSA,are involved in positive gene regulation. A newly identified region -745 to -705 bp of GCLC gene,which can be bound by USF,is involved in negative gene regulation,suggesting that the interaction between E-box and USF can inhibit the expression of γ-GCS.