Delineation of the insulin-responsive sequence in the rat cytosolic aspartate aminotransferase gene: binding sites for hepatocyte nuclear factor-3 and nuclear factor I.
Expression of the rat cytosolic aspartate aminotransferase gene is stimulated by glucocorticoids and repressed by insulin in the liver. The regulation by insulin and part of the glucocorticoid effect are mediated by a distal region in the promoter. A 142 bp fragment (-1844 to -1702) confers hormonal sensitivity to the heterologous thymidine kinase promoter in transient-transfection assays in H4IIEC3 hepatoma cells. Footprinting and gel-shift assays showed that several nuclear proteins bind to this region at conserved CCAAT-enhancer binding protein (C/EBP), activator protein (AP-1) and E-box sequences. Hepatocyte nuclear factor-3alpha (HNF-3)alpha and beta bind to sequences upstream of a glucocorticoid-responsive element (GRE) half-site as demonstrated by supershift experiments. Nuclear factor I (NFI)-like proteins bind downstream of the GRE half-site. These sites around the GRE motif overlap with five insulin responsive element (IRE) -like sequences (TG/ATTT). The effect of insulin was not prevented by any single mutation in the IRE-like sites. However, mutation of two IRE sites (namely IREc and d) prevented the insulin effect although only marginally affecting the glucocorticoid effect. The results suggest that the effect of insulin is due to a complex interplay of factors requiring the synergistic contribution of at least two sites and underline the contribution of HNF-3 and NFI-like proteins.
Beurton
F
F
Unité INSERM 490, Université René Descartes, 45 Rue des Saints Pères, 75270 Paris cedex 06, France.
Bandyopadhyay
U
U
Dieumegard
B
B
Barouki
R
R
Aggerbeck
M
M
D000818:Animals; D001219:Aspartate Aminotransferases / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D051541:Hepatocyte Nuclear Factor 3-alpha; D051542:Hepatocyte Nuclear Factor 3-beta; D007328:Insulin / Q000494:pharmacology* / Q000502:physiology; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C496501:Foxa1 protein, rat; C496504:Foxa2 protein, rat; D005938:Glucocorticoids; D051541:Hepatocyte Nuclear Factor 3-alpha; D007328:Insulin; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D051542:Hepatocyte Nuclear Factor 3-beta; D001219:Aspartate Aminotransferases