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 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 HNF3beta or HNF3beta 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 HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta 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 HNF3beta 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.
Scassa
María E
ME
Laboratorio de Biología Molecular, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II Piso 4, Ciudad Universitaria, 1428 Buenos Aires, Argentina.
Guberman
Alejandra S
AS
Ceruti
Julieta M
JM
Cánepa
Eduardo T
ET
D000624:5-Aminolevulinate Synthetase / Q000096:biosynthesis*; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D005799:Genes, Dominant; D005822:Genetic Vectors; D006367:HeLa Cells; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C496502:FOXA2 protein, human; D007328:Insulin; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase; D000624:5-Aminolevulinate Synthetase