Functional analysis of rat liver citrate carrier promoter: differential responsiveness to polyunsaturated fatty acids

Abstract

CiC (citrate carrier), a mitochondrial membrane protein, plays an important metabolic role by transporting acetyl-CoA into the cytosol for fatty acid and cholesterol synthesis. Several studies showed that CiC activity and expression is regulated by dietary fatty acids. In the present study we report data on the structural and functional characterization of the 5'-flanking region of the rat Cic gene. By transient transfection assays in H4IIE rat hepatoma cells, a PUFA (polyunsaturated fatty acids) response region has been identified within the CiC promoter. A cluster of putative binding sites for several transcription factors, composed of a NF-Y (nuclear factor-Y) site, an E-box-like site, a SRE1 (sterol regulatory element 1)-like site and four Sp1 (stimulatory protein 1) sites, was localized in the promoter region. Luciferase reporter gene and gel mobility shift assays indicated that a functional E-box-like, essential to the basal CiC promoter activity, confers responsiveness to activation by SREBP (SRE-binding protein)-1c. This study provides evidence for SREBP-1c as a principal target for PUFA regulation of CiC transcription. In H4IIE cells, overexpression of nSREBP (nuclear SREBP)-1c over-rides arachidonic acid (C(20:4, n-6)) suppression, but does not prevent the repression by docosahexaenoic acid (C(22:6, n-3)). ChIP (chromatin immunoprecipitation) assays in H4IIE cells showed that docosahexaenoic acid affects the binding of NF-Y, Sp1 and SREBP-1 to the PUFA response region of CiC promoter, whereas arachidonic acid alters only the binding of SREBP-1. Our data show that PUFA inhibition of hepatic Cic gene transcription is mediated not only by the nuclear level of SREBP-1c, but also might involve a reduction in Sp1 and NF-Y DNA binding, suggesting differential mechanisms in the Cic gene regulation by different PUFA.