title: HNF-4α regulates expression of human ornithin carbamoyltransferase through interaction with two positive cis-acting regulatory elements located in the proximal promoter.
abstract: OTC encodes ornithine carbamoyltransferase, mitochondrial matrix enzyme involved in the synthesis of urea. The tissue-specific expression of OTC in the liver and intestine is dependent on the interaction of OTC promoter with an upstream enhancer. HNF-4 and C/EBPβ are crucial for this interaction in the rat and mouse. In the present study we focused on characterization of elements involved in the regulation of OTC transcription in human. Using a set of 5'-deleted promoter mutants in a reporter assay we identified two positive cis-acting regulatory elements located at c.-105 and c.-136 within the human OTC promoter. Both are essential for the transcriptional activity of the promoter itself and for the interaction with the enhancer. Protein binding at the corresponding sites was confirmed by DNase I footprinting. Electromobility shift assay with a specific competitor and anti-HNF-4α antibody identified the DNA-protein binding sites as HNF-4α recognition motifs. A third HNF-4α binding site has been found at the position c.-187. All three HNF-4α binding sites are located within 35 bp upstream of the transcription start sites at positions c.-95, c.-119 (major) and c.-169 (minor). A series of C/EBPβ recognition motifs was identified within the enhancer. Involvement of C/EBPβ and HNF-4α in the promoter-enhancer interaction is further supported by a massive DNAprotein interaction observed in the footprinting and EMSA assays. Since the OTC promoter lacks general core promoter elements such as TATA-box or initiators in standard positions, HNF-4α most likely plays an essential role in the initiation of OTC transcription in human.
authors: Laboratory of Experimental Hepatology, Institute for Clinical and Experimental Medicine and Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University in Prague, Czech Republic.
authors: O
authors: O
authors: Lukšan
authors: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic.
authors: L
authors: L
authors: Dvořáková
authors: Laboratory of Experimental Hepatology, Institute for Clinical and Experimental Medicine and Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University in Prague, Czech Republic.
authors: M
authors: M
authors: Jirsa
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009954:Ornithine Carbamoyltransferase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D016415:Sequence Alignment; D014158:Transcription, Genetic
chemical_list: C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D004247:DNA; D008156:Luciferases; D009954:Ornithine Carbamoyltransferase; D003850:Deoxyribonuclease I