Transcriptional regulatory regions for expression of the rat fatty acid synthase.
We previously mapped the sequences responsive to insulin/glucose stimulation and polyunsaturated fatty acid (PUFA) suppression in proximal promoter region from -57 to -35 of fatty acid synthase (FAS) gene of rat liver [Fukuda et al. (1996) Biochem. Mol. Biol. Int. 38, 987-9961. When two copies of the sequences spanning -57 to -35 were linked to a reporter gene containing heterologous promoter and were used for transfection, the reporter activity significantly increased in response to insulin/glucose treatment in hepetocytes. This increase was inhibited by addition of PUFA. Gel mobility shift assays using the sequence from -57 to -35 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 GC-rich region located within -57 to -35 of the FAS promoter. Cotransfection studies in rat hepatocytes, with the Sp1 expression vector and FAScat constructs, showed the inactivation of the promoter. These results were similar to those for the region from -68 to -52 of FAS gene (an insulin response element). The region from -68 to -52 of FAS gene competed for the formation of DNA-protein complexes to the region from -57 to -35 in the gel shift assay. Mutational analysis showed that the overlapping region of these two sequences was essential for the binding of Sp1. It has been demonstrated that both the regions from -57 to -35 and from -68 to -52 of the FAS gene are responsible for regulation due to insulin/glucose and PUFA, and Sp1 may be involved in the regulation.