2 ADJACENT C/EBP-BINDING SEQUENCES THAT PARTICIPATE IN THE CELL-SPECIFIC EXPRESSION OF THE MOUSE SERUM AMYLOID A3 GENE
2 ADJACENT C/EBP-BINDING SEQUENCES THAT PARTICIPATE IN THE CELL-SPECIFIC EXPRESSION OF THE MOUSE SERUM AMYLOID A3 GENE
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DOI：
10.1128/mcb.10.12.6624
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发表时间：
1990-12-01
影响因子：
5.3
通讯作者：
LIAO, WSL
中科院分区：
文献类型：
--
作者：
LI, XX;HUANG, JH;LIAO, WSL
血清淀粉样蛋白A(SAA)是一种主要在肝脏合成的急性期蛋白。它在正常动物中的表达很低，但在急性炎症后会增加数百倍。为了检测参与肝脏特异性表达的DNA序列，通过瞬时转染、带移和DNase I保护实验分析了小鼠SAA3基因的5‘-’侧翼区。我们发现，在TATA盒上立即有一个5‘端的56bp片段，跨越相对于转录起始点的-93到-38区域，足以以一种剂量依赖和方向无关的方式将肝细胞特异性转录激活赋予异源启动子。该DNA片段可以与热稳定的核蛋白形成DNA-蛋白质复合体，形成的复合体可以被与C/EBP或DBP结合部位对应的过量低聚物特异性竞争，而不是与其他三种肝脏特异性因子HNF1、HNF3和HNF4的结合部位竞争。使用Hep3B核提取物进行的足迹分析显示，在这段56bp的片段中有两个相邻的足迹区域，远端区域的亲和力至少是近端区域的五倍。当使用纯化的重组C/EBP蛋白时，观察到相同的足迹模式。这些结果表明，C/EBP与该56bp片段的结合在体内对增强小鼠SAA3基因在肝细胞中的表达起着重要作用。
Serum amyloid A (SAA) is a major acute-phase protein synthesized primarily in the liver. Its expression, very low in normal animals, is increased several hundredfold following acute inflammation. To examine DNA sequences involved in liver-specific expression, 5''-flanking regions of the mouse SAA3 gene were analyzed by transient transfection, bandshift, and DNase I protection assays. We found that a 56-bp fragment immediately 5'' to the TATA box spanning the region -93 to -38 relative to the transcription start site was sufficient to confer liver cell-specific transcriptional activation onto a heterologous promoter in a dose-dependent and orientation-independent manner. This DNA fragment could form DNA-protein complexes with heat-stable nuclear proteins, and the complexes formed could be specifically competed for by excess oligomers corresponding to the C/EBP- or DBP-binding sites but not by binding sites for three other liver-specific factors, HNF1, HNF3, and HNF4. Footprint analysis using Hep3B nuclear extracts revealed two adjacent footprint regions within this 56-bp fragment, the distal region having at least fivefold-greater affinity than the proximal region. Identical footprint patterns were observed when purified recombinant C/EBP protein was used. These results indicated that binding of C/EBP to this 56-bp fragment plays an important role in vivo in enhancing expression of the mouse SAA3 gene in hepatocytes.