TRANSCRIPTIONAL REGULATION OF THE RAT FATTY-ACID SYNTHASE GENE - IDENTIFICATION AND FUNCTIONAL-ANALYSIS OF POSITIVE AND NEGATIVE EFFECTORS OF BASAL TRANSCRIPTION

The gene for fatty acid synthase (FAS), which contains both GC-rich se quences and a TATA box in its promoter region, is expressed in a tissu e-specific manner in response to developmental, nutritional and hormon al signals. Here we report the identification of sequence elements in the 5'-flanking region responsible for modulation of basal promoter ac tivity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 pr eadipocytes with plasmids containing the chloroamphenicol acetyltransf erase gene driven by FAS promoter sequences of different lengths revea led that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprin ting, electrophoretic mobility-shift assays and mutagenesis. The seque nce element is a typical GC box and the nuclear protein binding to thi s region appears immunochemically indistinguishable from Spl. The seco nd positive regulatory element, an inverted CCAAT box, was localized t o nucleotides -98/-92 by electrophoretic mobility-shift assays and mut agenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucl eotides -319 and -301 by DNase I footprinting, electrophoretic mobilit y-shift assays and deletion mutagenesis; this region consists of 78%, G residues. In conclusion, initiation of FAS transcription from a sing le start site is enhanced by the presence of an adjacent TATA motif, a n inverted CCAAT box and an upstream binding site for the transcriptio n factor Spl; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream n egative regulatory element.