Abstract
As the sole protein component of low density lipoproteins, apolipoprotein B (apoB) plays an important role in cholesterol metabolism. Previously, we found that the proximal promoter region of apoB (-81 to -52 relative to the start site) played a critical role in hepatocyte-specific gene expression and that that region contained overlapping binding sites for nuclear factors AF-1 (-81 to -62) and C/EBP (-69 to -52) (Metzger, S., Leff, T., and Breslow, J. L. (1990) J. Biol. Chem. 265, 9978-9983). In this study, we show that HNF-4, a member of the steroid hormone receptor superfamily, binds the AF-1 site on the apoB promoter and 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 correlation of HNF-4 binding and transcriptional activity. In addition, transient co-transfection experiments with HNF-4 and C/EBP alpha expression vectors showed that the two factors can synergistically activate transcription to levels more than 3-fold above the sum of either factor alone. Finally, using gel retardation analysis we show that purified HNF-4 and C/EBP proteins can concurrently occupy their overlapping binding sites on the apoB promoter in vitro. However, since the same system showed a lack of cooperative binding, we argue that an alternative mechanism is responsible for the synergistic effect of HNF-4 and C/EBP alpha on apoB gene transcription.
Highlights
As the sole protein component of low density lipopro- coronary heart disease (Sniderman et al, 1980; Brunzell et teins, apolipoprotein B plays an important role al., 1984), while moderately decreased levels of LDL cholesin cholesterol metabolism
B-48 is using gel retardation analysis we show that purified HNF-4 and C E B P proteins can concurrently occupy their overlapping binding sites on the apolipoprotein B (apoB) promoter in vitro
We show that HNF-4 is quantitatively the major factor in liver nuclear extracts interacting with the AF-1 binding site in theapoB gene
Summary
HNF-4, acloned transcriptionfactor of the steroid hormone from rat liver nuclear extract by the method of Sladek et aZ. (1990). Crude rat liver nuclear extract was passed over a heparinagarosecolumn followedby a double-stranded DNA-cellulosecolumn. Both columns were developed with salt gradients and the fractions assayed for activity by a gel retardation assay using the 20-base pair C/EBP oligo. We show that HNF-4 is quantitatively the major factor in liver nuclear extracts interacting with the AF-1 binding site in theapoB gene. We show that HNF-4 and This material, which showed several bands on a silver-stained gel, C/EBPa synergistically activate transcription of the apoB promoter. Conditions for DNA binding reactions with crude nuclear extracts and purified HNF-4 and C/EBPproteins are given in the legends to Figs.
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