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, 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.
Metzger
S
S
Laboratory of Biochemical Genetics and Metabolism, Rockefeller University, New York, New York 10021.
Halaas
J L
JL
Breslow
J L
JL
Sladek
F M
FM
D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D001055:Apolipoproteins B; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D014157:Transcription Factors; D004247:DNA