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Abstract:

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/EBPα or C/EBPβ expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPα and C/EBPβ. Co-transfection with C/EBPα or C/EBPβ expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPα or C/EBPβ still increased the promoter activity of both pC/EBP(mut)-116/+36p8-C4T (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/EBPα and C/EBPβ 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.

Registro:

Documento: Artículo
Título:Structural and functional characterization of the mouse p8 gene: Promotion of transcription by the CAAT-enhancer binding protein α (C/EBPα) and C/EBPβ trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter
Autor:Vasseur, S.; Mallo, G.V.; Garcia-Montero, A.; Ortiz, E.M.; Fiedler, F.; Cánepa, E.; Moreno, S.; Iovanna, J.L.
Filiación:U.315 INSERM, 46 Boulevard de la Gaye, F-13009 Marseille, France
Institut für Anästhesie, Klinikum Mannheim, Theodor-Kutzer-Ufer, D-68167, Mannheim, Germany
Depto. de Quim. Biológica, Fac. de Ciencias Exactas y Naturales, Ciudad Universitaria-Pabellon 2, Argentina
Palabras clave:3T3 cells; CAT activity; Gene expression; antibody; chloramphenicol acetyltransferase; cis acting element; DNA; DNA binding protein; protein; protein p8; trans acting factor; unclassified drug; animal cell; article; cell strain 3t3; consensus sequence; controlled study; DNA flanking region; DNA protein complex; expression vector; gel mobility shift assay; gene; gene structure; mouse; nonhuman; nucleotide sequence; oligonucleotide probe; priority journal; promoter region; reporter gene; sequence homology; site directed mutagenesis; structure activity relation; transcription regulation; 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Basic Helix-Loop-Helix Transcription Factors; Binding Sites; CCAAT-Enhancer-Binding Proteins; Cloning, Molecular; Consensus Sequence; DNA; DNA-Binding Proteins; Exons; Expressed Sequence Tags; Genes, Reporter; Growth Substances; Humans; Introns; Mice; Molecular Sequence Data; Neoplasm Proteins; Nuclear Proteins; Promoter Regions (Genetics); Response Elements; Sequence Alignment; Sequence Deletion; Trans-Activation (Genetics); Animalia; Felis catus; Rodentia
Año:1999
Volumen:343
Número:2
Página de inicio:377
Página de fin:383
DOI: http://dx.doi.org/10.1042/0264-6021:3430377
Título revista:Biochemical Journal
Título revista abreviado:Biochem. J.
ISSN:02646021
CODEN:BIJOA
CAS:Basic Helix-Loop-Helix Transcription Factors; CCAAT-Enhancer-Binding Proteins; DNA, 9007-49-2; DNA-Binding Proteins; Growth Substances; Neoplasm Proteins; Nuclear Proteins; Nupr1 protein, mouse; P8 protein, human
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v343_n2_p377_Vasseur

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Citas:

---------- APA ----------
Vasseur, S., Mallo, G.V., Garcia-Montero, A., Ortiz, E.M., Fiedler, F., Cánepa, E., Moreno, S.,..., Iovanna, J.L. (1999) . Structural and functional characterization of the mouse p8 gene: Promotion of transcription by the CAAT-enhancer binding protein α (C/EBPα) and C/EBPβ trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter. Biochemical Journal, 343(2), 377-383.
http://dx.doi.org/10.1042/0264-6021:3430377
---------- CHICAGO ----------
Vasseur, S., Mallo, G.V., Garcia-Montero, A., Ortiz, E.M., Fiedler, F., Cánepa, E., et al. "Structural and functional characterization of the mouse p8 gene: Promotion of transcription by the CAAT-enhancer binding protein α (C/EBPα) and C/EBPβ trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter" . Biochemical Journal 343, no. 2 (1999) : 377-383.
http://dx.doi.org/10.1042/0264-6021:3430377
---------- MLA ----------
Vasseur, S., Mallo, G.V., Garcia-Montero, A., Ortiz, E.M., Fiedler, F., Cánepa, E., et al. "Structural and functional characterization of the mouse p8 gene: Promotion of transcription by the CAAT-enhancer binding protein α (C/EBPα) and C/EBPβ trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter" . Biochemical Journal, vol. 343, no. 2, 1999, pp. 377-383.
http://dx.doi.org/10.1042/0264-6021:3430377
---------- VANCOUVER ----------
Vasseur, S., Mallo, G.V., Garcia-Montero, A., Ortiz, E.M., Fiedler, F., Cánepa, E., et al. Structural and functional characterization of the mouse p8 gene: Promotion of transcription by the CAAT-enhancer binding protein α (C/EBPα) and C/EBPβ trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter. Biochem. J. 1999;343(2):377-383.
http://dx.doi.org/10.1042/0264-6021:3430377