Orphan receptor HNF-4 and bZip protein C/EBP alpha bind to overlapping regions of the apolipoprotein B gene promoter and synergistically activate transcription

• Published in: The Journal of biological chemistry 1993-08, Vol.268 (22), p.16831-16838
• Main Authors: Metzger, S, Halaas, J.L, Breslow, J.L, Sladek, F.M
• Format: Article
• Language: English
• Subjects: ADN; Apolipoproteins B - genetics; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Biological and medical sciences; CCAAT-Enhancer-Binding Proteins; Cell Line; DNA; DNA-Binding Proteins - metabolism; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; GENERO HUMANO; GENETICA; GENETIQUE; GENRE HUMAIN; Hepatocyte Nuclear Factor 4; Humans; LIPOPROTEINAS; LIPOPROTEINE; Liver - metabolism; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nuclear Proteins - metabolism; Phosphoproteins; Promoter Regions, Genetic; Protein Binding; PROTEINAS; PROTEINE; SECUENCIA NUCLEICA; SEQUENCE NUCLEIQUE; Transcription Factors - metabolism; Transcription. Transcription factor. Splicing. Rna processing; Transcriptional Activation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)85491-1

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-l/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/EBPalpha on apoB gene transcription