DISTAL APOLIPOPROTEIN-C-III REGULATORY ELEMENT-F TO ELEMENT-J ACT AS A GENERAL MODULAR ENHANCER FOR PROXIMAL PROMOTERS THAT CONTAIN HORMONERESPONSE ELEMENTS - SYNERGISM BETWEEN HEPATIC NUCLEAR FACTOR-IV MOLECULES BOUND TO THE PROXIMAL PROMOTER AND DISTAL ENHANCER SITES

Transient transfection assays have shown that the distal apoC-III prom oter segments that contain the regulatory elements F to J enhance the strength of the tandemly linked proximal apoA-I promoter 5- to 13-fold in hepatic (HepG2) cells. Activation in intestinal (CaCo-2) cells to levels comparable to those obtained in HepG2 cells requires a larger a poA-I promoter sequence that extends to nucleotide -1500 as well as th e presence of hepatic nuclear factor-4 (HNF-4). The distal apoC-III re gulatory elements can also enhance 4- to 8-fold the strength of the he terologous apoB promoter in HepG2 and CaCo-2 cells. Finally, these ele ments in the presence of HNF-4 enhance 14.5- to 18.5-fold the strength of the minimal adenovirus major late promoter linked to two copies of the hormone response element (HRE) AID of apoA-I in both HepG2 and Ca Co-2 cells. In vitro mutagenesis of the promoter/enhancer cluster esta blished that the enhancer activity is lost by a mutation in the HRE pr esent in the 3' end of the regulatory element I (-736 to -714) and is reduced significantly by point mutations or deletions in one or more o f the regulatory elements F to J of the apoC-III enhancer. The enhance r activity also requires the HREs of the proximal apoA-I promoter. The apoC-III enhancer can also restore the activity of the proximal apoA- I and apoB promoters that have been inactivated by mutations in CCAAT/ enhancer binding protein binding sites, indicating that C/EBP may not participate in the synergistic activation of the promoter/enhancer clu ster. The findings suggest that the regulatory elements F to J of the apoC-III promoter act as a general modular enhancer that can potentiat e the strength of proximal promoters that contain HREs. Such potentiat ion in the HepG2 cells can be accounted for by synergistic interaction s between HNF-4 or Ether nuclear hormone receptors bound to the proxim al and distal HREs and SP1 or ether factors bound to the apoC-III enha ncer. Additional factors may be required for optimal activity in CaCo- 2 cells as well as for the function of this region as an intestinal en hancer.