Structural and functional characterization of the mouse p8 gene: promotion of transcription by the CAAT-enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter.
Rat p8 mRNA was discovered because of its strong activation in pancreas during the acute phase of pancreatitis. We report here structural and functional data on the mouse p8 gene. The mouse p8 polypeptide is 80 amino acids long and shows 91% and 75% identity with its rat and human counterparts respectively. The p8 gene is organized into three exons interrupted by two introns. Promoter regions involved in the regulation of p8 gene expression in NIH 3T3 cells were analysed. Chloramphenicol acetyltransferase (CAT) reporter assays with progressive deletions of the 5' flanking region of the mouse p8 gene revealed four silencer elements located from nucleotides -5000 to -1472, -1471 to -671, -670 to -473, and -239 to -117 respectively. One positive element was identified between nucleotides -117 and -72. We identified a CAAT-enhancer binding protein (C/EBP) cis-acting element at position -111. Site-directed mutagenesis of this consensus site decreased promoter activity to 5% of that of the wild-type. An electrophoretic mobility-shift assay, using an oligonucleotide probe corresponding to the C/EBP consensus and nuclear extracts of NIH 3T3 cells transfected with C/EBPalpha or C/EBPbeta expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPalpha and C/EBPbeta. Co-transfection with C/EBPalpha or C/EBPbeta expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPalpha or C/EBPbeta still increased the promoter activity of both pC/EBPmut-116/+36p8-CAT (which contains the C/EBP mutated site) and the p-71/+36-CAT construct (which does not contain the C/EBP site). Collectively, these results show that C/EBPalpha and C/EBPbeta trans-acting factors can promote transcription of the mouse p8 gene (i) by direct binding to the C/EBP consensus site, and (ii) by enhancing the activity of other trans-acting factors interacting with the p8 promoter.
Vasseur
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U.315 INSERM, 46 Boulevard de la Gaye, F-13009 Marseille, France.
Mallo
G V
GV
Garcia-Montero
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Ortiz
E M
EM
Fiedler
F
F
Cánepa
E
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Moreno
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Iovanna
J L
JL
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D003001:Cloning, Molecular; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D020224:Expressed Sequence Tags; D017930:Genes, Reporter / Q000235:genetics; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020218:Response Elements / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D015533:Transcriptional Activation
D051792:Basic Helix-Loop-Helix Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D006133:Growth Substances; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C491643:NUPR1 protein, human; C491644:Nupr1 protein, mouse