McGlynn L K, Mueller C R, Begbie M, Notley C R, Lillicrap D
Department of Pathology, Queen's University, Kingston, Ontario, Canada.
Mol Cell Biol. 1996 May;16(5):1936-45. doi: 10.1128/MCB.16.5.1936.
Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5' untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) approximately 300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates approximately 1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide -44 results in a significant but small increase in promoter activity. The activity of each of the various 5' deletion constructs is significantly enhanced by cotransfection of C/EBPalpha and D-site-binding protein expression plasmids, while cotransfection of both C/EBPalpha and C/EBPbeta plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides -740 and -1002. Since the minimal promoter sequence (-44 to +148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5' untranslated region.
凝血因子VIII是正常止血功能所需的一种重要辅助因子。因子VIII缺乏会导致出血性疾病甲型血友病。尽管因子VIII基因在十年前就已被克隆，但控制其转录的机制仍未明确。在我们的研究中，我们通过用大鼠肝核提取物进行的DNase I保护试验，对因子VIII启动子内的12个蛋白质结合位点进行了表征。其中三个元件（位点1至3）位于基因的5'非翻译区内，而另外三个位点（位点4至6）位于转录起始位点上游的前100 bp内。我们在位点6上游约300 bp处鉴定出另一个位点（位点7），以及在一个250 bp区域内的五个位点簇，该区域在距转录起始位点约1 kb处终止。这些结合位点中的七个（位点2、3、4、6、7、9和10）结合转录因子C/EBP家族的成员。DBP也结合其中五个位点（位点3、4、6、7和9）。利用在HepG2细胞中的瞬时转染研究，我们表明缺失核苷酸-44远端的因子VIII启动子序列会导致启动子活性显著但小幅增加。通过共转染C/EBPα和D位点结合蛋白表达质粒，各种5'缺失构建体的活性均显著增强，而共转染C/EBPα和C/EBPβ质粒则导致反式激活进一步增强。这些研究还提供了位于核苷酸-740和-1002之间的阻遏元件的证据。由于最小启动子序列（-44至+148）维持全长启动子序列的转录活性，我们接着鉴定与位点1至4结合的其他因子。竞争研究表明，一种普遍存在的转录因子NF-Y结合位点4，而肝脏富集转录因子肝细胞核因子I（HNF-1）结合位点1。最小启动子的突变分析表明，HNF-1对于体外激活因子VIII基因的转录至关重要。我们的结果还表明，我们鉴定出的多个上游元件可能在5'非翻译区内的HNF-元件被破坏时作为备用调控区域起作用。
