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 sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5'-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed 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 footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.