Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression
Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthas...
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| Autores principales: | , , , |
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| Formato: | Artículo publishedVersion |
| Lenguaje: | Inglés |
| Publicado: |
2004
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| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_00219258_v279_n27_p28082_Scassa |
| Aporte de: |
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paperaa:paper_00219258_v279_n27_p28082_Scassa |
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| record_format |
dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| language |
Inglés |
| orig_language_str_mv |
eng |
| topic |
Bioassay Genes Insulin Molecular biology Physiology Gene expressions Hepatic nuclear factors Molecular mechanisms Enzymes 5 aminolevulinate synthase adenosine triphosphate antisense oligodeoxynucleotide chloramphenicol acetyltransferase complementary DNA cyclic AMP responsive element binding protein hepatic nuclear factor 3 insulin messenger RNA nuclear factor I phosphoenolpyruvate carboxykinase (GTP) phosphorus 32 protein kinase B transcription factor unclassified drug article cyclic AMP responsive element gene expression regulation genetic transcription hepatoma cell hormonal regulation human human cell insulin responsive element nucleotide sequence plasmid priority journal promoter region 5-Aminolevulinate Synthetase Base Sequence Binding Sites Blotting, Southern Blotting, Western CCAAT-Enhancer-Binding Proteins Cell Line Cell Line, Tumor Cell Nucleus Chloramphenicol O-Acetyltransferase DNA-Binding Proteins Enzyme Inhibitors Gene Deletion Gene Expression Regulation, Enzymologic Genes, Dominant Genetic Vectors Hela Cells Hepatocyte Nuclear Factor 3-beta Humans Insulin Molecular Sequence Data Mutation NFI Transcription Factors Nuclear Proteins Oligonucleotides, Antisense Phosphorylation Plasmids Promoter Regions (Genetics) RNA RNA, Messenger Trans-Activation (Genetics) Transcription Factors Transcription, Genetic Transfection |
| spellingShingle |
Bioassay Genes Insulin Molecular biology Physiology Gene expressions Hepatic nuclear factors Molecular mechanisms Enzymes 5 aminolevulinate synthase adenosine triphosphate antisense oligodeoxynucleotide chloramphenicol acetyltransferase complementary DNA cyclic AMP responsive element binding protein hepatic nuclear factor 3 insulin messenger RNA nuclear factor I phosphoenolpyruvate carboxykinase (GTP) phosphorus 32 protein kinase B transcription factor unclassified drug article cyclic AMP responsive element gene expression regulation genetic transcription hepatoma cell hormonal regulation human human cell insulin responsive element nucleotide sequence plasmid priority journal promoter region 5-Aminolevulinate Synthetase Base Sequence Binding Sites Blotting, Southern Blotting, Western CCAAT-Enhancer-Binding Proteins Cell Line Cell Line, Tumor Cell Nucleus Chloramphenicol O-Acetyltransferase DNA-Binding Proteins Enzyme Inhibitors Gene Deletion Gene Expression Regulation, Enzymologic Genes, Dominant Genetic Vectors Hela Cells Hepatocyte Nuclear Factor 3-beta Humans Insulin Molecular Sequence Data Mutation NFI Transcription Factors Nuclear Proteins Oligonucleotides, Antisense Phosphorylation Plasmids Promoter Regions (Genetics) RNA RNA, Messenger Trans-Activation (Genetics) Transcription Factors Transcription, Genetic Transfection Scassa, M.E. Guberman, A.S. Ceruti, J.M. Cánepa, E.T. Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression |
| topic_facet |
Bioassay Genes Insulin Molecular biology Physiology Gene expressions Hepatic nuclear factors Molecular mechanisms Enzymes 5 aminolevulinate synthase adenosine triphosphate antisense oligodeoxynucleotide chloramphenicol acetyltransferase complementary DNA cyclic AMP responsive element binding protein hepatic nuclear factor 3 insulin messenger RNA nuclear factor I phosphoenolpyruvate carboxykinase (GTP) phosphorus 32 protein kinase B transcription factor unclassified drug article cyclic AMP responsive element gene expression regulation genetic transcription hepatoma cell hormonal regulation human human cell insulin responsive element nucleotide sequence plasmid priority journal promoter region 5-Aminolevulinate Synthetase Base Sequence Binding Sites Blotting, Southern Blotting, Western CCAAT-Enhancer-Binding Proteins Cell Line Cell Line, Tumor Cell Nucleus Chloramphenicol O-Acetyltransferase DNA-Binding Proteins Enzyme Inhibitors Gene Deletion Gene Expression Regulation, Enzymologic Genes, Dominant Genetic Vectors Hela Cells Hepatocyte Nuclear Factor 3-beta Humans Insulin Molecular Sequence Data Mutation NFI Transcription Factors Nuclear Proteins Oligonucleotides, Antisense Phosphorylation Plasmids Promoter Regions (Genetics) RNA RNA, Messenger Trans-Activation (Genetics) Transcription Factors Transcription, Genetic Transfection |
| description |
Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5′-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3β or HNF3β plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3β is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3β can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3β binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters. |
| format |
Artículo Artículo publishedVersion |
| author |
Scassa, M.E. Guberman, A.S. Ceruti, J.M. Cánepa, E.T. |
| author_facet |
Scassa, M.E. Guberman, A.S. Ceruti, J.M. Cánepa, E.T. |
| author_sort |
Scassa, M.E. |
| title |
Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression |
| title_short |
Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression |
| title_full |
Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression |
| title_fullStr |
Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression |
| title_full_unstemmed |
Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression |
| title_sort |
hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression |
| publishDate |
2004 |
| url |
http://hdl.handle.net/20.500.12110/paper_00219258_v279_n27_p28082_Scassa |
| work_keys_str_mv |
AT scassame hepaticnuclearfactor3andnuclearfactor1regulate5aminolevulinatesynthasegeneexpressionandareinvolvedininsulinrepression AT gubermanas hepaticnuclearfactor3andnuclearfactor1regulate5aminolevulinatesynthasegeneexpressionandareinvolvedininsulinrepression AT cerutijm hepaticnuclearfactor3andnuclearfactor1regulate5aminolevulinatesynthasegeneexpressionandareinvolvedininsulinrepression AT canepaet hepaticnuclearfactor3andnuclearfactor1regulate5aminolevulinatesynthasegeneexpressionandareinvolvedininsulinrepression |
| _version_ |
1769810014351917056 |
| spelling |
paperaa:paper_00219258_v279_n27_p28082_Scassa2023-06-12T16:42:51Z Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression J. Biol. Chem. 2004;279(27):28082-28092 Scassa, M.E. Guberman, A.S. Ceruti, J.M. Cánepa, E.T. Bioassay Genes Insulin Molecular biology Physiology Gene expressions Hepatic nuclear factors Molecular mechanisms Enzymes 5 aminolevulinate synthase adenosine triphosphate antisense oligodeoxynucleotide chloramphenicol acetyltransferase complementary DNA cyclic AMP responsive element binding protein hepatic nuclear factor 3 insulin messenger RNA nuclear factor I phosphoenolpyruvate carboxykinase (GTP) phosphorus 32 protein kinase B transcription factor unclassified drug article cyclic AMP responsive element gene expression regulation genetic transcription hepatoma cell hormonal regulation human human cell insulin responsive element nucleotide sequence plasmid priority journal promoter region 5-Aminolevulinate Synthetase Base Sequence Binding Sites Blotting, Southern Blotting, Western CCAAT-Enhancer-Binding Proteins Cell Line Cell Line, Tumor Cell Nucleus Chloramphenicol O-Acetyltransferase DNA-Binding Proteins Enzyme Inhibitors Gene Deletion Gene Expression Regulation, Enzymologic Genes, Dominant Genetic Vectors Hela Cells Hepatocyte Nuclear Factor 3-beta Humans Insulin Molecular Sequence Data Mutation NFI Transcription Factors Nuclear Proteins Oligonucleotides, Antisense Phosphorylation Plasmids Promoter Regions (Genetics) RNA RNA, Messenger Trans-Activation (Genetics) Transcription Factors Transcription, Genetic Transfection Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5′-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3β or HNF3β plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3β is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3β can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3β binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters. Fil:Scassa, M.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Guberman, A.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ceruti, J.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Cánepa, E.T. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2004 info:eu-repo/semantics/article info:ar-repo/semantics/artículo info:eu-repo/semantics/publishedVersion application/pdf eng info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00219258_v279_n27_p28082_Scassa |