摘要
目的确定XBP1启动子的转录核心区域,探讨激活转录因子6(ATF6)在内质网应激(ERS)与非ERS状态下对XBP1启动子转录活性的调控作用。方法采用基因重组技术构建ATF6基因及两个缺失体ATF6s1p、ATF6s2p的真核表达载体pcDNA3.1(-)-ATF6、pcDNA3.1(-)-s1p、pcDNA3.1(-)-s2p,以及XBP1启动子报告基因载体pGL3-XBP1。针对XBP1启动子的各个组成元件设计和构建XBP1一系列截短体的报告基因载体。采用双荧光素酶报告基因法检测并确定XBP1启动子的核心启动子区,Western blotting检测ATF6及缺失体s2p、s1p在ERS与非ERS状态下对XBP1核心启动子区转录的调控作用。结果 XBP1启动子的-407bp-+133bp区域是转录活性调控的核心区域,-2000bp--407bp区域不具有转录活性,该区域可能含有抑制转录活性的组成元件。非ERS状态时,ATF6及两个缺失体s2p、s1p可明显上调XBP1启动子的转录活性及XBP1蛋白表达;而在ERS状态时,ATF6及两个缺失体s2p、s1p下调了XBP1启动子的转录活性和XBP1蛋白表达。结论 ATF6对XBP1的转录调控作用在ERS状态和非ERS状态下存在差异。非ERS状态时,ATF6上调XBP1的转录和表达,而ERS状态时,ATF6下调XBP1的转录和表达。
Objective To determine the transcriptional core area of the XBP1 promoter and the effect on XBP1 transcription activity by ATF6 in endoplasmic reticulum stress（ERS） or non-ERS.Methods pcDNA3.1（-）-ATF6,the eukaryotic expression vector of ATF6 gene,and pcDNA3.1（-）-s1p and pcDNA3.1（-）-s2p,the vectors for deletion mutants of ATF6 gene（ATF6s1p and ATF6s2p）,and pGL3-XBP1,the XBP1 reporter gene vector were constructed with gene recombination technology.A series of reporter gene vectors of the truncated XBP1 promoter were designed and constructed based on every component element of the XBP1 promoter.The transcription core area of XBP1 promoter was detected and determined by the dual-luciferase reporter assay.The effect on transcription activity of XBP1 core promoter by ATF6 and its two deletion mutants in ERS or non-ERS were then analyzed with Western blotting.Results The region from-407bp to ＋133bp of XBP1 promoter was the core area.However,the region from-2000bp to-407bp did not exhibit any transcription activity.This finding indicated that the region from-2000bp to-407bp might contain element-inhibiting transcription activity.In the non-ERS model,the transcription activity of ATF6 and the two deletion mutants s2p and s1p to XBP1 promoter were notably higher than those of the negative control.Meanwhile,Western blotting revealed that XBP1 expression also increased after transfection with ATF6 and its two deletion mutants.In ERS,the transcription activity of ATF6 and the two deletion mutants s2p and s1p to XBP1 promoter notably decreased.Accordingly,XBP1 expression exhibited reduction after transfection.Conclusions XBP1 transcription regulated by ATF6 displayed a significant difference in ERS or non-ERS models.The transcription and expression of XBP1 were upregulated by ATF6 during non-ERS,but were downregulated by ATF6 during ERS.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2012年第4期317-321,共5页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(31040019
81171697)~~
关键词
激活转录因子6
基因表达调控
转录启动子
activating transcription factor 6
gene expression regulation
transcription initiation site
