Transcriptional regulatory region for expression of the rat ATP citrate-lyase gene.
We previously mapped the sequences responsive to insulin/glucose stimulation and polyunsaturated fatty-acid suppression in the proximal promoter region between positions -104 and -20 of the ATP citrate-lyase (ACL) gene [Fukuda, H., Iritani, N., Katsurada, A. & Noguchi, T. (1996) FEBS Lett. 380, 204-207]. To investigate further the regulatory DNA sequences required for stimulation and suppression of this gene, primary cultured hepatocytes were transfected with plasmids containing the 5'-flanking sequences of the rat ACL gene fused to the chloramphenicol acetyltransferase (CAT) gene. When two copies of the sequences spanning -64 to -41 (linked to ACLcat20) were used for transfection, CAT activity significantly increased in response to insulin/glucose treatment. This increase was inhibited by addition of polyunsaturated fatty acid. Mutational analysis of this region showed that sequences between -55 and -51 are essential for recognition and interaction with trans-acting factors. Gel mobility shift assays using the sequence from -64 to -41 as a probe revealed nuclear factor(s) from rat liver that specifically complexed with the sequences. In addition, by antibody supershift assays, we have detected the binding of the transcriptional factor Sp1 at the G+C-rich region located within -64 to -41 of the ACL promoter. On the other hand, the formations of DNA-protein complexes with Sp1 binding site or ACL(-64 to -41) were decreased in rats fed a high-carbohydrate diet in comparison with those in rats fasted or fed a polyunsaturated fatty-acid-rich diet. Cotransfection studies in rat hepatocytes, with the Sp1 expression vector and ACLcat constructs, showed the inactivation of the promoter. These results demonstrated that the region from -64 to -41 of the ACL gene was responsible for stimulation due to insulin/glucose, the stimulation was suppressed by polyunsaturated fatty acid, and Sp1 may be involved in the regulation.
Fukuda
H
H
Tezukayama Gakuin College, Osaka, Japan.
Iritani
N
N
Noguchi
T
T
D001275:ATP Citrate (pro-S)-Lyase / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D008099:Liver / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis; D008297:Male; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D001275:ATP Citrate (pro-S)-Lyase