HNF-4α regulates expression of human ornithin carbamoyltransferase through interaction with two positive cis-acting regulatory elements located in the proximal promoter
Language English Country Czech Republic Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
25056436
DOI
10.14712/fb2014060030133
PII: file/5725/fb2014a0017.pdf
Knihovny.cz E-resources
- MeSH
- 5' Flanking Region genetics MeSH
- Hep G2 Cells MeSH
- Deoxyribonuclease I metabolism MeSH
- DNA Footprinting MeSH
- DNA metabolism MeSH
- Transcription, Genetic MeSH
- Hepatocyte Nuclear Factor 4 metabolism MeSH
- Rats MeSH
- Humans MeSH
- Luciferases metabolism MeSH
- Molecular Sequence Data MeSH
- Mutation genetics MeSH
- Mice MeSH
- Ornithine Carbamoyltransferase genetics metabolism MeSH
- Computer Simulation MeSH
- Promoter Regions, Genetic * MeSH
- Gene Expression Regulation, Enzymologic * MeSH
- Genes, Reporter MeSH
- Electrophoretic Mobility Shift Assay MeSH
- Base Sequence MeSH
- Sequence Alignment MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Deoxyribonuclease I MeSH
- DNA MeSH
- Hepatocyte Nuclear Factor 4 MeSH
- HNF4A protein, human MeSH Browser
- Luciferases MeSH
- Ornithine Carbamoyltransferase MeSH
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.
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