In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer.
In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer.
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使用直接基因转移体内鉴定小鼠肾素基因中的负调控元件。
DOI：
10.1172/jci118156
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Dzau,VJ
中科院分区：
文献类型：
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作者：
Yamada,T;Horiuchi,M;Morishita,R;Zhang,L;Pratt,RE;Dzau,VJ
DBA/2 J小鼠含有两个肾素基因位点（Ren 1d和Ren 2d）。Ren 2d在下颌下腺（SMG）中表达，而Ren 1d则不表达，而两者均在肾脏中表达。基于体外研究，我们推测，在肾素基因启动子负调控元件（NRE）参与其组织特异性表达。在这项研究中，我们研究了在体内水平上使用直接基因转移的分子机制。将Ren 1d或Ren 2d启动子片段与氯霉素乙酰转移酶（CAT）基因表达载体融合。将这些复合在融合脂质体中的构建体直接注射到小鼠SMG中或通过肾动脉动脉内注射到小鼠肾脏中。含有CAT的载体在SMG和肾脏中表现出容易检测的体内表达。在SMG中，含有NRE的Ren 1d片段废除了CAT表达，而缺失NRE则恢复了CAT表达。来自Ren 2d启动子的同源片段不抑制CAT表达，而缺失150-bp插入导致抑制。共转染Ren 1d构建体与Ren 1d-NRE寡核苷酸作为转录因子诱饵恢复CAT表达。与SMG相反，用Ren 1d片段-CAT构建体或Ren 2d片段-CAT构建体转染肾脏导致相似的CAT表达水平。有趣的是，人c-myc NRE寡核苷酸，共享与Ren 1d-NRE的同源性有效地竞争与这些寡核苷酸的Ren 1d基因的表达在体内的调节。该NRE序列也与多种哺乳动物基因中发现的沉默元件同源，表明存在一个NRE/NRE结合蛋白家族，调节不同基因的表达。图片
DBA/2J mouse contains two renin gene loci (Ren1d and Ren2d). Ren2d but not Ren1d is expressed in submandibular gland (SMG) while both are expressed in the kidney. Based on vitro studies, we have postulated that a negative regulatory element (NRE) in the renin gene promoter is involved in its tissue-specific expression. In this study, we examined the molecular mechanism at the in vivo level using direct gene transfer. Fragments of the Ren1d or Ren2d promoter were fused to a chloramphenicol acetyltransferase (CAT) gene expression vector. These constructs complexed in fusogenic liposomes were injected directly into the mouse SMG or intraarterially into the mouse kidney via the renal artery. The vector containing the CAT exhibited readily detectable in vivo expressions in both SMG and kidney. In the SMG, Ren1d fragment containing the NRE abolished CAT expression while deletion of the NRE restored CAT expression. The homologous fragment from the Ren2d promoter did not inhibit CAT expression while deletion of the 150-bp insertion resulted in the inhibition. Cotransfection of Ren1d construct with Ren1d-NRE oligonucleotides as transcriptional factor decoy restored CAT expression. Contrary to the SMG, transfection with Ren1d fragment-CAT construct or Ren2d fragment-CAT construct into the kidney resulted in similar levels of CAT expression. Interestingly, human c-myc NRE oligonucleotides which share homology with Ren1d-NRE competed effectively with these oligonucleotides for the regulation of Ren1d gene expression in vivo. This NRE sequence is also homologous to silencer elements found in multiple mammalian genes, suggesting the presence of a family of NRE/NRE binding proteins regulating expression of diverse genes.Images