Characterization of tissue-specific enhancer elements in the second intron of the human apolipoprotein B gene.
We report the identification and characterization of tissue-specific transcriptional enhancer elements that influence the expression of the human apolipoprotein B gene. A 704-base pair PstI fragment comprising sequences from the first and second introns of the human apolipoprotein B gene (positions +360 to +1064) possesses tissue-specific transcriptional enhancer elements when assayed in transient transfection experiments using either the apolipoprotein B or thymidine kinase promoter. The majority of the enhancer activity, which was observed in transcriptionally active HepG2 and CaCo-2 cells, but not in transcriptionally inactive Chinese hamster ovary or HeLa cells, was subsequently localized to a 443-base pair SmaI-PvuII fragment (positions +621 to +1064) within the second intron of the apolipoprotein B gene. Gel retention experiments demonstrated that sequence motifs within this region interact with a number of nuclear proteins from HepG2, CaCo-2, and HeLa cells. The actual sequence elements that bound to nuclear proteins from HepG2 cells were identified by DNase I footprinting. Deletion experiments were performed to distinguish those protein-binding regions involved in the enhancer effect. Our data demonstrate that sequences between positions +806 and +940 are essential for this enhancer activity. This segment contains one large 97-base pair footprint, whose sequence has been conserved between the human and mouse genes. Binding sites for the liver-specific transcription factors HNF-1 and HNF-3 are present within this footprint.
Brooks
A R
AR
Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, San Francisco, California 94140-0608.
Blackhart
B D
BD
Haubold
K
K
Levy-Wilson
B
B
D001055:Apolipoproteins B / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D016172:DNA Fingerprinting; D004587:Electrophoresis, Agar Gel; D004742:Enhancer Elements, Genetic; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010957:Plasmids; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D001055:Apolipoproteins B; D009687:Nuclear Proteins