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 ce lls, suggesting a tissue specific transcriptional regulation of its ge ne. To investigate the mechanism controlling apo A-IV transcription we have analysed its promoter region by in vitro DNA binding and transie nt transfection experiments. DNase I footprinting analysis of the prox imal 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 maxim al promoter activity, binds HNF-4, Arp-1 and Ear-3 with similar affini ty in a mutually exclusive manner. HNF-4 transactivated chimeric const ructs containing intact AIVC site in the context of either the apo A-I V promoter or the heterologous thymidine kinase minimal promoter, whil e 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 f actors for the same recognition site. In transient transfection assays the apo A-IV promoter region (- 700 to + 10) had a very law activity in cells of hepatic (HepG2) and intestinal (CaCo2) origin. This activi ty was increased 13 to 18-fold when the upstream elements of the dista ntly linked apo C-III gene were fused to the proximal promoter. Result s 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 e nhancement in HepG2 and CaCo2 cells. The apo C-III upstream elements e nhanced 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 c onstructs containing single HNF-4 sites. The findings suggest that the enhancer effect is mediated by synergistic interactions between the t rans-acting factors which recognize the apo C-III regulatory elements and HNF-4 which binds to the proximal apo A-IV promoter.