cis-acting elements and transcription factors involved in the promoter activity of the human factor VIII gene.
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 VIII gene.
Figueiredo
M S
MS
Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
Brownlee
G G
GG
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005169:Factor VIII / Q000096:biosynthesis / Q000235:genetics*; D005796:Genes; D017930:Genes, Reporter; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000473:pathology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D016328:NF-kappa B; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005169:Factor VIII; D008156:Luciferases