Transcriptional regulation of the apolipoprotein A-IV gene involves synergism between a proximal orphan receptor response element and a distant enhancer located in the upstream promoter region of the apolipoprotein C-III gene.
Apolipoprotein A-IV expression is limited to intestinal and hepatic cells, suggesting a tissue specific transcriptional regulation of its gene. To investigate the mechanism controlling apo A-IV transcription we have analysed its promoter region by in vitro DNA binding and transient transfection experiments. DNase I footprinting analysis of the proximal promoter with rat liver nuclear extracts revealed four protected regions: AIVA (-32 to -22), AIVB (-84 to -42), AIVC (-148 to -92) and AIVD (-274 to -250). Element AIVC which is necessary for maximal promoter activity, binds HNF-4, Arp-1 and Ear-3 with similar affinity in a mutually exclusive manner. HNF-4 transactivated chimeric constructs containing intact AIVC site in the context of either the apo A-IV promoter or the heterologous thymidine kinase minimal promoter, while Arp-1 and Ear-3 repressed this activation. Increasing amounts of HNF-4 alleviated Arp-1 or Ear-3 mediated repression, suggesting that the observed opposing effects is a result of direct competition of these factors for the same recognition site. In transient transfection assays the apo A-IV promoter region (-700 to +10) had a very low activity in cells of hepatic (HepG2) and intestinal (CaCo2) origin. This activity was increased 13 to 18-fold when the upstream elements of the distantly linked apo C-III gene were fused to the proximal promoter. Results obtained with different 5' and 3' deletion constructs indicated that the cis-acting elements F to J between the nucleotides -500 and -890 of the apo C-III promoter were absolutely necessary to drive maximal enhancement in HepG2 and CaCo2 cells. The apo C-III upstream elements enhanced the activity of the minimal AdML promoter or the apo A-IV site C mutant less efficiently than the intact apo A-IV or AdML promoter constructs containing single HNF-4 sites. The findings suggest that the enhancer effect is mediated by synergistic interactions between the trans-acting factors which recognize the apo C-III regulatory elements and HNF-4 which binds to the proximal apo A-IV promoter.
Ktistaki
E
E
Institute of Molecular Biology and Biotechnology, University of Crete Medical School, Greece.
Lacorte
J M
JM
Katrakili
N
N
Zannis
V I
VI
Talianidis
I
I
D000818:Animals; D053305:Apolipoprotein C-III; D001054:Apolipoproteins A / Q000235:genetics*; D001056:Apolipoproteins C / Q000235:genetics*; D001483:Base Sequence; D051838:COUP Transcription Factor I; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D051557:Hepatocyte Nuclear Factor 4; D007422:Intestines / Q000166:cytology; D008099:Liver / Q000737:chemistry / Q000166:cytology; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010750:Phosphoproteins; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D011987:Receptors, Steroid / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D053305:Apolipoprotein C-III; D001054:Apolipoproteins A; D001056:Apolipoproteins C; D051838:COUP Transcription Factor I; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C497591:Nr2f1 protein, rat; C497588:Nr2f2 protein, rat; D010750:Phosphoproteins; D011987:Receptors, Steroid; D015534:Trans-Activators; D014157:Transcription Factors; C022205:apolipoprotein A-IV