ORPHAN RECEPTOR HNF-4 AND BZIP PROTEIN C EBP-ALPHA BIND TO OVERLAPPING REGIONS OF THE APOLIPOPROTEIN-B GENE PROMOTER AND SYNERGISTICALLY ACTIVATE TRANSCRIPTION/

As the sole protein component of low density lipoproteins, apolipoprot ein B (apoB) plays an important role in cholesterol metabolism. Previo usly, we found that the proximal promoter region of apoB (-81 to -52 r elative to the start site) played a critical role in hepatocyte-specif ic gene expression and that that region contained overlapping binding sites for nuclear factors AF-1 (-81 to -62) and C/EBP (-69 to -52) (Me tzger, S., Leff, T., and Breslow, J. L. (1990) J. Biol. Chem. 265, 997 8-9983). In this study, we show that HNF-4, a member of the steroid ho rmone receptor superfamily, binds the AF-1 site on the apoB promoter a nd through it activates transcription in transient transfection assays in both liver and non-liver cell lines, HepG2 and HeLa, respectively. Mutational analysis of the AF-1/HNF-4 binding site indicated a correl ation of HNF-4 binding and transcriptional activity. In addition, tran sient co-transfection experiments with HNF-4 and C/EBPalpha expression vectors showed that the two factors can synergistically activate tran scription to levels more than 3-fold above the sum of either factor al one. Finally, using gel retardation analysis we show that purified HNF -4 and C/EBP proteins can concurrently occupy their overlapping bindin g sites on the apoB promoter in vitro. However, since the same system showed a lack of cooperative binding, we argue that an alternative mec hanism is responsible for the synergistic effect of HNF-4 and C/EBPalp ha on apoB gene transcription.