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
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 respe...
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1999
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v343_n2_p377_Vasseur http://hdl.handle.net/20.500.12110/paper_02646021_v343_n2_p377_Vasseur |
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paper:paper_02646021_v343_n2_p377_Vasseur2023-06-08T15:23:11Z 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 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 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. 1999 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v343_n2_p377_Vasseur http://hdl.handle.net/20.500.12110/paper_02646021_v343_n2_p377_Vasseur |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 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 |
| topic_facet |
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 |
| description |
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. |
| title |
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 |
| title_short |
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 |
| title_full |
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 |
| title_fullStr |
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 |
| title_full_unstemmed |
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 |
| title_sort |
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 |
| publishDate |
1999 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_02646021_v343_n2_p377_Vasseur http://hdl.handle.net/20.500.12110/paper_02646021_v343_n2_p377_Vasseur |
| _version_ |
1768541607081017344 |