title: In vivo functional characterization of the aldolase B gene enhancer.
abstract: A 400-bp intronic enhancer fragment in conjunction with the proximal promoter of the aldolase B gene provided correct tissue-specific expression in transgenic mice together with hormonal regulation in the liver. We investigated in vivo and in cultured cells the contribution of the intronic regulatory sequences and their interaction with the promoter elements in controlling aldolase B gene expression. Transgene activity was completely abolished by disruption of the two hepatocyte nuclear factor 1 (HNF1) binding sites in the enhancer, whereas mutation of one HNF1 site had no effect in the liver but strongly decreased activity in the kidney. Our data show that the HNF1 binding site(s) in the enhancer were key regulators of aldolase B transgene expression both in the liver and kidney. Deletion of the CCAAT/enhancer-binding protein site in the promoter completely abolished the enhancer function in HepG2 cells. These results suggest that expression of the aldolase B gene in the liver requires cooperative interactions between CCAAT/enhancer-binding protein and HNF1. Deletion of the HNF4 binding site in the enhancer suppressed expression in both liver and kidney in half of the transgenic lines, suggesting that this element might play a role in chromatin opening at the insertion site. We firmly establish that the endogenous aldolase B gene's first response to glucagon or cyclic AMP exposure was a transient increase in the expression in the liver, followed by a secondary decline in the transcription, as previously reported. This response was reproduced by all transgenes studied, indicating that neither HNF1 nor HNF4 binding sites in the enhancer were involved in this biphasic cyclic AMP response.
authors.affiliation: Département de Génétique, Développement et Pathologie Moléculaire, Institut Cochin, INSERM, CNRS et Université René Descartes, Paris 75014, France.
authors.forename: Claudine
authors.initials: C
authors.lastname: Gregori
authors.forename: Arlette
authors.initials: A
authors.lastname: Porteu
authors.forename: Claudia
authors.initials: C
authors.lastname: Mitchell
authors.forename: Axel
authors.initials: A
authors.lastname: Kahn
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Pichard
mesh_terms: D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D015152:Blotting, Northern; D003994:Bucladesine / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics*; D017353:Gene Deletion; D005934:Glucagon / Q000378:metabolism / Q000494:pharmacology; D051557:Hepatocyte Nuclear Factor 4; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D019076:Transgenes
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; C501970:Tcfl4 protein, mouse; D014157:Transcription Factors; D012313:RNA; D003994:Bucladesine; D005934:Glucagon; D005634:Fructose-Bisphosphate Aldolase