The mechanism by which the human apolipoprotein B gene reducer operates involves blocking of transcriptional activation by hepatocyte nuclear factor 3.
Previously, we showed that when a DNA fragment extending from -3067 to -2734 of the human apolipoprotein B (apo-B) gene is inserted immediately upstream of an apo-B promoter segment (-139 to +121), transcription from this promoter is reduced by about 10-fold in cultured colon carcinoma cells (CaCo-2) but not in cultured hepatoma cells (HepG2). We postulated that this reducer operates by a mechanism involving active repression of a transcriptional activator that binds to the segment from -111 to -88 of the apo-B promoter (B. Paulweber and B. Levy-Wilson, J. Biol. Chem. 266:24161-24168 1991). In the current study, the reducer element has been localized to a 24-bp sequence from -2801 to -2778 of the apo-B gene that contains a binding site for the negative regulatory protein ARP-1. Furthermore, we have demonstrated that the transcription factor hepatocyte nuclear factor 3 alpha (HNF-3 alpha) binds to the sequence 5'-TGTTTGCTTTTC-3' from -95 to -106 of the apo-B promoter, to stimulate transcription. Transcriptional activation by HNF-3 is repressed when the reducer sequence is inserted immediately upstream of the HNF-3 binding site, suggesting a mechanism by which the reducer-bound protein blocks the activation promoted by HNF-3. Data from cotransfection experiments in which ARP-1 is overexpressed in the absence of its binding site suggest that ARP-1 interacts either directly or via a mediator protein with proteins recognizing the HNF-3 site and that this interaction is sufficient to repress transcriptional activation by HNF-3. Because transcriptional activation by Sp1 is not affected by the reducer, it is unlikely that the reducer interacts directly with basic components of the transcriptional machinery.
Paulweber
B
B
First Department of Internal Medicine, Landeskrankenanstalten Salzburg, Austria.
Sandhofer
F
F
Levy-Wilson
B
B
D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D003106:Colon / Q000166:cytology; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D051541:Hepatocyte Nuclear Factor 3-alpha; D006801:Humans; D008099:Liver / Q000166:cytology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism / Q000494:pharmacology*; D009841:Oligonucleotides / Q000378:metabolism; D011987:Receptors, Steroid; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*; D014407:Tumor Cells, Cultured
D001055:Apolipoproteins B; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011987:Receptors, Steroid; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C496499:FOXA1 protein, human; C095569:NR2F2 protein, human