Identification and characterization of a 315-base pair enhancer, located more than 55 kilobases 5 ' of the apolipoprotein B gene, that confers expression in the intestine

We recently reported that an 8-kilobase (kb) region, spanning from -54 to - 62 kb 5' of the human apolipoprotein B (apoB) gene, contains intestine-spec ific regulatory elements that control apoB expression in the intestines of transgenic mice. In this study, we further localized the apoB intestinal co ntrol region to a 3-kb segment (-54 to -57 kb). DNaseI hypersensitivity stu dies uncovered a prominent DNaseI hypersensitivity site, located within a 3 15-base pair (bp) fragment at the 5'-end of the 3-kb segment, in transcript ionally active CaCo-2 cells but not in transcriptionally inactive HeLa cell s. Transient transfection experiments with CaCo-2 and HepG2 cells indicated that the 315-bp fragment contained an intestine-specific enhancer, and ana lysis of the DNA sequence revealed putative binding sites for the tissue-sp ecific transcription factors hepatocyte nuclear factor 3 beta, hepatocyte n uclear factor 4, and CAAT enhancer-binding protein beta. Binding of these f actors to the 315-bp enhancer was demonstrated in gel retardation experimen ts. Transfection of deletion mutants of the 315-bp enhancer revealed the re lative contributions of these transcription factors in the activity of the apoB intestinal enhancer. The corresponding segment of the mouse apoB gene (located -40 to -83 kb 5' of the structural gene) exhibited a high degree o f sequence conservation in the binding sites for the key transcriptional ac tivators and also exhibited enhancer activity in transient transfection ass ays with CaCo-2 cells. In transgenic mouse expression studies, the 315-bp e nhancer conferred intestinal expression to human apoB transgenes.