We have mapped the DNase I-and micrococcal nuclease-hypersensitive sites present in the 5' end of the human apolipoprotein B (apo-B) gene in nuclei from cells expressing or not expressing the gene. Four DNase I-hypersensitive sites were found in nuclei from liver-derived HepG2 cells and intestine-derived CaCo-2 cells, which express the apo-B gene, but not in HeLa cells, which do not. These sites are located near positions -120, -440, -700, and +760 Apolipoprotein B (apo-B) is the major protein component of low-density lipoproteins, which play a central role in the metabolism and transport of cholesterol. Apo-B is the ligand responsible for the uptake and clearance of low-density lipoproteins from the circulation via the apo-B,E(LDL) receptor pathway (13,20,29,30). We and others have recently determined the primary structure of apo-B by sequencing its cDNA (5, 7, 24, 26), and we have elucidated the complete structure of the 43-kilobase (kb) human apo-B gene (3). The gene comprises 29 exons and 28 introns, with most introns appearing in the 5'-terminal one-third of the gene. Apo-B mRNA has been detected only in the liver and intestines of several mammals, including humans (23), demonstrating that transcription of the apo-B gene is regulated in a tissue-specific manner.To understand the molecular mechanisms involved in the tissue-specific expression of the human apo-B gene, we have begun to map cis-acting regulatory sequences in the 5' flanking region of the gene. We also have examined this region for the presence of DNase I-hypersensitive (DH) sites in cells expressing or not expressing the gene. These sites are located near the 5' ends of the genes that are active or inducible in the cell under study, and their presence often correlates with binding sites for regulatory proteins (for a review, see reference 9). We have also examined the susceptibility of the 5' end of the apo-B gene to another nuclease, micrococcal nuclease (MNase). This enzyme differs from DNase I in its mode of nucleolytic cleavage (22) and thus provides complementary structural information.In the experiments in this study, we show that a 1-kb PvuII fragment from the 5' end of the gene exhibits cellspecific promoter activity. Furthermore, several DH and MNase-hypersensitive (MH) sites were detected in the 5' proximal region of the human apo-B gene in human hepatoma HepG2 cells and colon carcinoma CaCo-2 cells. These sites are absent from cell types that do not express the gene, * Corresponding author.such as HeLa cells. Most of these sites map to regions in which highly conserved base sequence motifs occur.
MATERIALS AND METHODSTissue culture. HepG2 cells were grown in monolayer in T150 flasks (Coming) in a CO2 incubator at 37°C in minimum essential medium supplemented with 10% fetal bovine serum. HeLa cells were grown in Dulbecco modified Eagle medium in the presence of 10% fetal calf serum. All cultures were supplemented with 1% penicillin-streptomycin.Undifferentiated CaCo-2 cells were grown as described above, but with 15% serum, and wer...