Nuclease-hypersensitive sites define a region with enhancer activity in the third intron of the human apolipoprotein B gene.
The positions of several DNase I-hypersensitive (DH) sites have been mapped in the second and third introns of the human apolipoprotein B gene. Two such DH sites, I and V, are present both in human hepatoma (HepG2) and colon carcinoma (CaCo-2) cells that express the gene but absent from HeLa cells that do not express the gene. These DH sites map near sequence elements that have been highly conserved between the human and mouse genes. A PvuII-EcoRI fragment (+1064 to +2977) from the hypersensitive region exhibited enhancer activity, which was further localized by means of deletion experiments to a 155-base pair segment located entirely within the third intron and flanked by two DH sites. Three DNase I footprints were observed within this core enhancer, one of which contains putative binding sites for three liver specific nuclear proteins. Experiments are presented that suggest that this enhancer operates by a similar mechanism as that described previously for the strong second intron enhancer, involving an interaction with the basal transcriptional machinery. Digestions with low levels of micrococcal nuclease were performed to ascertain whether nucleosomes were present in the DNase I sensitive enhancer region. Nine different micrococcal nuclease-hypersensitive (MH) sites were detected in HepG2 cells but not in HeLa cells; one MH site was common to both cell types, and HeLa cells exhibited three unique MH sites. The first six MH sites (I-VI) are spaced approximately 200 base pairs apart, suggesting the presence of positioned nucleosomes in that region. MH sites VI-X are more closely spaced, suggesting either additional cutting sites within the core particle or the absence of one or two nucleosomes in this segment of the third intron enhancer.
Levy-Wilson
B
B
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100.
Paulweber
B
B
Nagy
B P
BP
Ludwig
E H
EH
Brooks
A R
AR
D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D016172:DNA Fingerprinting; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006801:Humans; D007438:Introns; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D001055:Apolipoproteins B; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease