IDENTIFICATION OF AN INVERTED CCAAT BOX MOTIF IN THE FATTY-ACID SYNTHASE GENE AS AN ESSENTIAL ELEMENT FOR MEDIATION OF TRANSCRIPTIONAL REGULATION BY CAMP

The antagonistic effect of cAMP on the insulin-induced expression of f atty acid synthase (FAS) in liver could be mimicked in vitro using H4I IE hepatoma cells, both by measuring the response of the endogenous FA S gene and by assaying expression of transfected reporter genes contai ning promoter elements of the FAS gene. 5'-Deletion analysis and repla cement mutagenesis revealed that an essential element required for cAM P antagonism of the insulin effect is an inverted CCAAT box located be tween nucleotides -99 and -92. DNase I footprinting and gel shift anal ysis revealed that this region can bind a protein present in nuclei of liver and spleen, organs that express high and undetectable levels of FAS, respectively. This protein is not a CCAAT/enhancer-binding prote in, C/EBP. Thus, the FAS gene appears unusual in that the sequence ele ment required for transcriptional regulation by cAMP is neither a cAMP response element (CRE) nor a binding site for AP-1, AP-2, or C/EBP. T hese results suggest that essential to the regulation of FAS transcrip tion by cAMP is the interaction of an inverted CCAAT box motif with a constitutively produced trans-acting factor that either itself undergo es modification in response to cAMP or associates with a protein that is produced or modified by cAMP exposure.