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Human Apolipoprotein B Gene Intestinal Control Region

机译：人载脂蛋白B基因肠道控制区

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摘要

Recently, we reported that a 315 bp enhancer, located over 55 kilobases (kb) upstream of the transcriptional start site of the human apolipoprotein B (apoB) gene, was sufficient to direct high-Ievel expression of human apoB transgenes in mice. In this report, we expand our analysis of the distant apoB intestinal control region (ICR), by examining the function of segments in the vicinity of the 315 bp intestinal enhancer (315 lE). DNaseI hypersensitivity (DH) studies of a 4.8 kb segment from the ICR revealed three new DH sites, in addition to the previously described DH1 region present within the 315 lE. DH2 mapped to a 485 bp segment (485 lE) immediately upstream of the 315 lE that exhibited strong intestinal enhancer activity in transient transfection experiments with intestine-derived CaCo-2 cells. Within the DH2 region, an HNF-4/ARP-1 binding site was demonstrated by gel retardation experiments. A 1.8 kb segment incorporating the 485 IE was capable of driving expression of human apoB transgenes in the intestines of mice. Additionally, a third component of the apoB ICR was found about 1.2 kb downstream of the 315 IE, within a 1031 bp segment (1031 lE) that also harbored two DH sites, DH3 and DH4. This segment did not display enhancer activity but was capable of driving transgene expression in the intestine. The three components of the ICR displayed a similar pattern of apoB mRNA expression along the horizontal axis of the intestine. The previously characterized in vivo liver-specific elements of the apoB gene, namely, the second intron enhancer and the 5' upstream liver enhancer, did not playa role in intestinal expression of apoB transgenes in mice.
机译：最近，我们报道了一个315 bp的增强子，位于人类载脂蛋白B（apoB）基因转录起始位点上游超过55千个碱基（kb）处，足以指导人类apoB转基因在小鼠中的高伊维尔表达。在本报告中，我们通过检查315 bp肠道增强子（315 lE）附近区段的功能，扩展了对远距离apoB肠道控制区（ICR）的分析。对来自ICR的4.8 kb片段的DNaseI超敏性（DH）研究揭示了三个新的DH位点，除了315 lE中存在的先前描述的DH1区。 DH2定位在紧接315 lE上游的485 bp片段（485 lE）上，在肠道来源的CaCo-2细胞的瞬时转染实验中，DH2表现出较强的肠道增强活性。在DH2区域内，通过凝胶阻滞实验证明了HNF-4 / ARP-1结合位点。结合了485 IE的1.8kb片段能够驱动人apoB转基因在小鼠肠中的表达。此外，发现apoB ICR的第三个成分位于315 IE下游约1.2 kb，位于1031 bp片段（1031 E）中，该片段也包含两个DH位点DH3和DH4。该区段不显示增强子活性，但是能够驱动肠中的转基因表达。 ICR的三个组成部分沿肠的水平轴显示出类似的apoB mRNA表达模式。先前表征的apoB基因的体内肝脏特异性元件，即第二内含子增强子和5'上游肝脏增强子，在小鼠apoB转基因肠表达中不起作用。

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