Huang Xiuli, Pawliczak Rafal, Yao Xiang-Lan, Madara Patricia, Alsaaty Sura, Shelhamer James H, Cowan Mark J
Critical Care Medicine Department, The National Institutes of Health, Bethesda, MD 20892, USA.
Gene. 2003 May 22;310:133-42. doi: 10.1016/s0378-1119(03)00529-8.
p11 is expressed in many different cell types, and serves a variety of regulatory functions. In order to better understand the transcriptional control of this protein, the 5' promoter region of the human p11 gene was cloned and sequenced. After confirming the transcription start point (TSP) using 5' rapid amplification of cDNA ends analysis, the 5' promoter was analysed. The sequence lacks a TATA box, but contains a variety of putative regulatory elements. There are two GAS sites, two AP-1 sites, two overlapping Sp-1 sites, and a gamma-IRE site clustered between -1080 and -1450. There is another cluster of putative regulatory sites between the TSP and -550 which contains two Sp-1 sites, two AP-2 sites, one GAS site, one NF-kappaB site, an incomplete CAAT box (8/9) and an overlapping Sp-1/AP-2 site at -17 to -26. Reporter gene constructs containing 4225 and 1498 bases 5' of the TSP demonstrated excellent unidirectional transcriptional activity in both constructs. Reporter genes containing serial 5' deletions were compared to the -1498 construct. The reporter gene which contained base pairs (bp) -36 to +89 had almost no activity. The reporter gene containing -188 to +89 had 50% of the -1498 construct, indicating that this sequence contains at least the minimal promoter. The Sp-1/AP-2 site near the transcription start site was studied by electrophoretic mobility shift and reporter gene assays. Addition of HeLa cell nuclear extract to labeled double-stranded (ds) oligonucleotide containing this sequence resulted in a gel shift which was inhibited by excess unlabeled ds oligonucleotide and by a consensus cold Sp-1 ds oligonucleotide, indicating specific Sp-1 binding. Excess AP-2 or NF-kappaB ds oligonucleotide had no effect on nuclear protein binding to the sequence. Mutation of the p11 wild-type Sp-1/AP-2 sequence eliminated both nuclear protein binding and the sequences ability to compete with native sequence for nuclear binding protein. A -1048 to +89 reporter construct containing a mutated Sp-1/AP-2 site resulted in a 40% decrease in transcriptional activity. Therefore, the 5' flanking sequence of the p11 gene exhibits promoter activity which may be localized to a variety of controlling regions, of which the proximal Sp-1/AP-2 site appears to be important for basal activity via its Sp-1 binding ability.
p11在许多不同的细胞类型中表达，并发挥多种调节功能。为了更好地理解该蛋白的转录调控，克隆并测序了人类p11基因的5'启动子区域。使用5' cDNA末端快速扩增分析确认转录起始点（TSP）后，对5'启动子进行了分析。该序列缺乏TATA盒，但包含多种假定的调控元件。在-1080至-1450之间聚集有两个GAS位点、两个AP-1位点、两个重叠的Sp-1位点和一个γ-IRE位点。在TSP和-550之间还有另一簇假定的调控位点，其中包含两个Sp-1位点、两个AP-2位点、一个GAS位点、一个NF-κB位点、一个不完整的CAAT盒（8/9）以及在-17至-26处的一个重叠的Sp-1/AP-2位点。包含TSP上游4225和1498个碱基的报告基因构建体在两种构建体中均表现出出色的单向转录活性。将含有连续5'缺失的报告基因与-1498构建体进行比较。包含碱基对（bp）-36至+89的报告基因几乎没有活性。包含-188至+89的报告基因具有-1498构建体50%的活性，表明该序列至少包含最小启动子。通过电泳迁移率变动分析和报告基因检测研究了转录起始位点附近的Sp-1/AP-2位点。将HeLa细胞核提取物添加到含有该序列的标记双链（ds）寡核苷酸中导致凝胶迁移，这被过量的未标记ds寡核苷酸和一致的冷Sp-1 ds寡核苷酸抑制，表明存在特异性Sp-1结合。过量的AP-2或NF-κB ds寡核苷酸对核蛋白与该序列的结合没有影响。p11野生型Sp-1/AP-2序列的突变消除了核蛋白结合以及该序列与天然序列竞争核结合蛋白的能力。含有突变Sp-1/AP-2位点的-1048至+89报告基因构建体导致转录活性降低40%。因此，p11基因的5'侧翼序列表现出启动子活性，其可能定位于多种控制区域，其中近端Sp-1/AP-2位点因其Sp-1结合能力似乎对基础活性很重要。
