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

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 idemified within the CiC promoter. A cluster of putative binding sites for several transcription factors, composed of a NF-Y (nuclear factor-Y) site, all E-box-like site, a SRE1 (sterol regulatory element 1)-like site and four Sp1 (stimulatory protein I) sites, was localized in the promoter region. Luciferase reporter gene and gel mobility shift assays indicated that a functional Ebox-like, essential to the basal CiC promoter activity, confers respensiveness to activation by SREBP (SRE-binding protein)-1c. This Study provides evidence for SREEIP-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,C-n-6) suppression, but (toes not prevent the repression by docosahexaenoic acid (C-22:6,C- n-3)-ChIP (chromatin immuno-precipitation) assays in H4IIE cells showed that docosahexaenoic acid affects the binding of NF-Y, Sp I and SREBP-I to the PUFA response region of CiC promoter, whereas arachidonic acid alters only the binding of SREB-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 ill Sp I and NF-Y DNA binding, Suggesting differential mechanisms in the Cic gene regulation by different PUFA.