Analysis by cell-free transcription of the liver-specific pyruvate kinase gene promoter.
A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor A1 (LF-A1), nuclear factor 1 (NF1), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNF1-binding site. In contrast, deletion of the HNF1-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.
Vaulont
S
S
Unité de Recherches en Génétique et Pathologie Moléculaires, INSERM U. 129-CHU Cochin, Paris, France.
Puzenat
N
N
Kahn
A
A
Raymondjean
M
M
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002467:Cell Nucleus / Q000032:analysis; D002474:Cell-Free System; D005934:Glucagon / Q000502:physiology; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009747:Nutritional Physiological Phenomena / Q000502:physiology; D009841:Oligonucleotides / Q000378:metabolism; D009928:Organ Specificity; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011770:Pyruvate Kinase / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic
D009841:Oligonucleotides; D014157:Transcription Factors; D005934:Glucagon; D011770:Pyruvate Kinase