Montgomery K T, Tardiff J, Reid L M, Krauter K S
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Mol Cell Biol. 1990 Jun;10(6):2625-37. doi: 10.1128/mcb.10.6.2625-2637.1990.
The alpha 1-protease inhibitor (alpha 1-PI) proteins of mice are encoded by a group of genes whose members are expressed coordinately in a liver-abundant pattern and are regulated primarily at the transcriptional level. To better understand the developmental and tissue-specific regulation of this gene family, one member that is analogous to the human alpha 1-antitrypsin gene was chosen for study. Deletional analysis of the upstream regulatory region of this gene was performed, spanning from -10 kilobases to -80 base pairs relative to the transcriptional start site. Two functional positive cis-acting elements within the 522 bases immediately upstream of the start site for transcription were shown to modulate the level of expression from this promoter when introduced into human or mouse hepatoma cells, and a third region acted as a negative regulatory element in that its deletion resulted in a two- to sixfold increase of expression of a transfected minigene construct. Sequence comparison between the regulatory domains of two mouse alpha 1-PI genes and the human alpha 1-antitrypsin gene showed that the mouse gene contains a novel positive cis-acting element which is absent in human gene and that a specific eight-base-pair difference between species results in a strong positive cis-acting element in the human gene acting as a negative element in the mouse gene. An enhancer located approximately 3,000 base pairs upstream of the major start site for transcription was also identified. This element is position and orientation independent. Several different DNA-protein binding assays were used to demonstrate that each DNA segment with functional significance in transfection assays interacts specifically with proteins found in adult mouse liver nuclei. The major positive-acting element appeared to be specifically recognized by nuclear proteins found only in tissues that express alpha 1-PI, while the negative element binding proteins were ubiquitous. Thus, the distal regulatory domain including bases -3500 to -133 of this murine alpha 1-PI gene family member is more complex than was previously demonstrated. It is composed of a set of at least three additional functional cis-acting regulatory elements besides those which have been mapped by others and has a far upstream enhancer.
小鼠的α1-蛋白酶抑制剂（α1-PI）蛋白由一组基因编码，其成员以肝脏丰富的模式协同表达，并且主要在转录水平上受到调控。为了更好地理解该基因家族的发育和组织特异性调控，选择了一个与人类α1-抗胰蛋白酶基因类似的成员进行研究。对该基因上游调控区域进行了缺失分析，范围从相对于转录起始位点的-10千碱基到-80碱基对。转录起始位点上游紧邻的522个碱基内的两个功能性正向顺式作用元件，当导入人或小鼠肝癌细胞时，可调节该启动子的表达水平，并且第三个区域作为负调控元件，其缺失导致转染的小基因构建体的表达增加两到六倍。两种小鼠α1-PI基因和人类α1-抗胰蛋白酶基因的调控域之间的序列比较表明小鼠基因含有一个人类基因中不存在的新型正向顺式作用元件，并且物种间特定的八个碱基对差异导致人类基因中的一个强正向顺式作用元件在小鼠基因中作为负向元件起作用。还鉴定了一个位于主要转录起始位点上游约3000碱基对处的增强子。该元件与位置和方向无关。使用了几种不同的DNA-蛋白质结合测定法来证明在转染测定中具有功能意义的每个DNA片段都与成年小鼠肝细胞核中发现的蛋白质特异性相互作用。主要的正向作用元件似乎仅被在表达α1-PI的组织中发现的核蛋白特异性识别，而负向元件结合蛋白则普遍存在。因此，该小鼠α1-PI基因家族成员的包括碱基-3500至-133的远端调控域比先前证明的更为复杂。它除了由其他人绘制图谱的那些元件外，还由一组至少三个额外的功能性顺式作用调控元件组成，并且有一个非常上游的增强子。
