CIS-ACTING ELEMENTS AND TRANSCRIPTION FACTORS INVOLVED IN THE PROMOTER ACTIVITY OF THE HUMAN FACTOR-VIII GENE
CIS-ACTING ELEMENTS AND TRANSCRIPTION FACTORS INVOLVED IN THE PROMOTER ACTIVITY OF THE HUMAN FACTOR-VIII GENE
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DOI：
10.1074/jbc.270.20.11828
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发表时间：
1995-05-19
影响因子：
4.8
通讯作者：
BROWNLEE, GG
中科院分区：
文献类型：
--
作者：
FIGUEIREDO, MS;BROWNLEE, GG
凝血因子VIII是一种对血液凝固至关重要的糖蛋白。尽管在多种人体组织中检测到因子VIII mRNA，但认为肝细胞是血浆因子VIII的主要来源。在这份报告中，我们证明了因子VIII基因的5 '-侧翼区域能够在三种人肝源性细胞系PLC/PRF/5，Chang和HepG 2中转录荧光素酶报告基因。DNA酶I足迹法显示存在19个蛋白质结合位点（标记为A至S，近端至远端），分布在因子VIII启动子的核苷酸-1175至-9区域（+1指翻译起始密码子，ATG）的沿着。在PLC/PRF/5细胞中对启动子区5'和3'缺失突变体的功能分析表明，从-279到-64的区域，包括位点B到D，包含最大启动子活性的所有必需元件。通过使用核提取物和纯化的转录因子的电泳迁移率变动分析，以及抗体超迁移分析，我们能够表征与近端启动子结合位点相互作用的四种肝脏富集因子和一种普遍存在的转录因子（部位A至E）：肝细胞核因子（HNF）1（位点A）、NF κ B（位点B）、C/EBP α和C/EBP β（位点C和位点D的近端和远端区域）和HNF 4（位点E）。另外，假定的TATA盒GATAAA（位置-201至-196）突变为GACCGA导致启动子活性降低不到2倍，表明假定的TATA盒对于因子VIII启动子活性不是必需的。这些结果显着有助于理解的控制因子MII基因的肝脏转录。
Factor VIII is a glycoprotein that is essential for blood coagulation. Although factor VIII mRNA has been detected in a variety of human tissues, hepatocytes are considered to be the major source of plasma factor VIII. In this report we demonstrate that the 5'-flanking region of the factor VIII gene is able to transcribe a luciferase reporter gene in three human liver-derived cell lines: PLC/PRF/5, Chang, and HepG2. DNase I footprinting showed the presence of 19 protein binding sites (labeled A to S, proximal to distal) distributed along the region from nucleotide -1175 to -9 of the factor VIII promoter (+1 refers to the translation initiation codon, ATG). Functional analysis of 5' and 3' deletion mutants of the promoter region in PLC/PRF/5 cells revealed that the region from -279 to -64, including sites B to D, contains all the necessary elements for maximal promoter activity. By using electrophoretic mobility shift assays with nuclear extracts and purified transcription factors, and antibody supershift assays we were able to characterize four liver-enriched factors and one ubiquitous transcription factor interacting with the proximal promoter binding sites (sites A to E): hepatocyte nuclear factor (HNF) 1 (site A), NF kappa B (site B), C/EBP alpha and C/EBP beta (proximal and distal regions of site C, and site D), and HNF4 (site E). Additionally, mutation of the putative TATA box GATAAA (positions -201 to -196) to GACCGA resulted in less than 2-fold decrease in promoter activity, suggesting that the putative TATA box is not essential for factor VIII promoter activity. These results significantly contribute to the understanding of the control of the hepatic transcription of the factor MII gene.