Antes T J, Goodart S A, Chen W, Levy-Wilson B
Research Institute, Palo Alto Medical Foundation, Palo Alto, California 94301, USA.
Biochemistry. 2001 Jun 12;40(23):6720-30. doi: 10.1021/bi010073a.
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-level 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 IE). 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 IE. DH2 mapped to a 485 bp segment (485 IE) immediately upstream of the 315 IE 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 IE) 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 play a role in intestinal expression of apoB transgenes in mice.
最近，我们报道了一个位于人类载脂蛋白B（apoB）基因转录起始位点上游超过55千碱基（kb）处的315 bp增强子，足以在小鼠中指导人类apoB转基因的高水平表达。在本报告中，我们通过研究315 bp肠道增强子（315 IE）附近片段的功能，扩展了对apoB远距离肠道控制区（ICR）的分析。对ICR中一个4.8 kb片段的DNaseI超敏反应（DH）研究揭示了三个新的DH位点，此外还有先前描述的位于315 IE内的DH1区域。DH2定位于315 IE上游紧邻的一个485 bp片段（485 IE），该片段在与肠道来源的CaCo-2细胞进行的瞬时转染实验中表现出强大的肠道增强子活性。在DH2区域内，凝胶阻滞实验证实了一个HNF-4/ARP-1结合位点。一个包含485 IE的1.8 kb片段能够在小鼠肠道中驱动人类apoB转基因的表达。此外，在315 IE下游约1.2 kb处，在一个1031 bp片段（1031 IE）内发现了apoB ICR的第三个组成部分，该片段也含有两个DH位点，DH3和DH4。该片段未显示增强子活性，但能够在肠道中驱动转基因表达。ICR的这三个组成部分在肠道横轴上显示出相似的apoB mRNA表达模式。apoB基因先前已表征的体内肝脏特异性元件，即第二个内含子增强子和5'上游肝脏增强子，在小鼠中apoB转基因的肠道表达中不起作用。
