Characterization of the human C6 promoter: requirement of the CCAAT enhancer binding protein binding site for C6 gene promoter activity.
The sixth complement component (C6) is a late-acting complement protein that participates in the assembly of the membrane attack complex. C6 and most of the complement proteins are mainly synthesized in the liver. However, the human hepatoma-derived cell line Hep-G2, which produces the majority of complement proteins, synthesizes traces of C6. Here, we have isolated and characterized the human C6 promoter. Approximately 1 kb of C6 upstream sequence is shown to be sufficient to achieve tissue-specific expression of a luciferase reporter gene in two hepatic (Hep-G2 and Hep-3B) and two extrahepatic cell lines (fibroblast M1 and HeLa) in a manner similar to endogenous C6. There are wide differences in C6 mRNA expression among the four cell lines, whereas Hep-3B expresses high levels of C6, Hep-G2 and M1 poorly synthesize C6, and HeLa completely lacks C6 expression. Deletional and mutational analysis demonstrates that a C/EBP (CCAAT/enhancer binding protein) site located at -67 is required for C6 expression in Hep-3B cells, but it has little effect in M1 and Hep-G2 cells. Electrophoretic mobility shift assays show that this sequence binds to a C/EBP alpha using Hep-3B nuclear extract, but a negligible activity is detected using a Hep-G2 extract. To further investigate whether C/EBP alpha is the limiting factor for C6 expression, we have transfected a C/EBP alpha expression vector into Hep-G2 and Ml cells. C/EBP alpha expression vector dramatically trans-activates the luciferase reporter gene controlled by the C6 promoter, and it partially restores C6 mRNA expression in Hep-G2 cells.
González
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S
Immunology Service, Central Hospital of Asturias, Oviedo, Spain.
López-Larrea
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C
D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000276:immunology; D003183:Complement C6 / Q000032:analysis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic / Q000276:immunology*; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic / Q000276:immunology*; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology; D015533:Transcriptional Activation; D014162:Transfection / Q000276:immunology; D014407:Tumor Cells, Cultured
D003183:Complement C6; D004268:DNA-Binding Proteins; D014157:Transcription Factors