Functional analysis of the endogenous retroviral promoter of the human endothelin B receptor gene
Functional analysis of the endogenous retroviral promoter of the human endothelin B receptor gene
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DOI：
10.1128/jvi.77.13.7459-7466.2003
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发表时间：
2003-07-01
影响因子：
5.4
通讯作者：
Mager, DL
中科院分区：
文献类型：
--
作者：
Landry, JR;Mager, DL
我们以前报道过，属于埃尔韦家族的逆转录病毒元件的长末端重复序列（LTR）通过提供替代启动子而促进人载脂蛋白C1（APOC 1）和内皮素B受体（EDNRB）基因的表达。虽然这两种LTR都显示出在体内和体外促进转录，但它们各自的活性和组织特异性似乎不同，即使它们具有高度的序列同一性。在本研究中，我们进一步表征了EDNRB LTR的启动子，并描绘了强活性所需的区域和基序。我们通过瞬时转染和定量实时PCR证实了LTR的胎盘限制性表达，并确定逆转录病毒启动子对胎盘中EDNRB转录物的水平有显著贡献，其中发现嵌合mRNA占总EDNRB mRNA的15%。在胎盘来源的细胞中瞬时转染5'缺失构建体鉴定了转录活性所必需的EDNRB LTR的位置111和122之间的基序，命名为LPE 1。去除这个区域，其中含有一个假定的SP1结合位点，废除启动子活性。第二个增强区位于LTR的位置175和215之间，称为LPE 2。有趣的是，由于微小的核苷酸差异，该部分包含APOC 1 LTR中不存在的三个结合位点。EDNRB LTR中的预测基序被发现可能在共生中起作用，因为对三个位点中的任何一个的修饰都会使转录减少三分之一，而对所有三个位点的改变都会消除启动子活性。本研究的结果说明了转录调控序列的微小变化如何对启动子活性产生深远的影响，并证明了人类内源性逆转录病毒元件对人类基因表达的复杂调控作用。
We previously reported that the long terminal repeats (LTRs) of retroviral elements belonging to the HERVE family contribute to the expression of the human apolipoprotein C1 (APOC1) and endothelin B receptor (EDNRB) genes by providing alternative promoters. While both LTRs were shown to promote transcription in vivo and in vitro, their respective activity and tissue specificity appeared to differ even though they shared a high degree of sequence identity. In the present study, we further characterized the promoter of the EDNRB LTR and delineated the regions and motifs required for strong activity. We confirmed the placenta-restricted expression of the LTR by transient transfections and quantitative real-time PCR and determined that the retroviral promoter contributes significantly to the level of EDNRB transcripts in placenta, where chimeric mRNAs were found to represent 15% of overall EDNRB mRNAs. Transient transfection of 5' deletion constructs in cells of placental origin identified a motif, named LPE1, between positions 111 and 122 of the EDNRB LTR necessary for transcriptional activity. Removal of this region, which contains a putative SP1 binding site, abolished promoter activity. A second enhancing region resides between positions 175 and 215 of the LTR and was termed LPE2. Interestingly, this section contained three binding sites that were not present in the APOC1 LTR due to minor nucleotide differences. The predicted motifs in the EDNRB LTR were found to likely act in symbiosis as modifications to any of the three sites reduced transcription by one-third while alterations to all three eliminated promoter activity. The results from this study illustrate how slight variations in transcriptional regulatory sequences can have a profound effect on promoter activity and demonstrate the complex regulatory effects of human endogenous retrovirus elements on human gene expression.