MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2653-2659
Vol. 10, No. 6
0270-7306/90/062653-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Characterization of the Promoter Elements Required for Hepatic and Intestinal Transcription of the Human apoB Gene: Definition of the DNA-Binding Site of a Tissue-Specific Transcriptional Factor DIMITRIS KARDASSIS,1 MARGARITA HADZOPOULOU-CLADARAS,1 DIPAK P. RAMJI,2 RICARDO CORTESE,2 VASSILIS I. ZANNIS,' AND CHRISTOS CLADARASl*
Section of Molecular Genetics, Cardiovascular Institute, Departments of Medicine and Biochemistry, Housman Medical Research Center, Boston University Medical Center, 80 East Concord Street, Boston, Massachusetts 02118,1 and The European Molecular Biology Laboratories, Meyerhofstrasse 1, 6900 Heidelberg, Federal Republic of Germany2 Received 11 September 1989/Accepted 16 February 1990
The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.
Apolipoprotein B is the sole protein component of lowdensity lipoprotein (LDL), constitutes 23.8% of the LDL particle (7, 12), and is involved in the recognition and catabolism of LDL by the LDL receptor (7, 8). The primary structure of human apoB-100 has been determined by derivation of cDNA and genomic and protein sequences (for a review, see reference 27). In humans, apoB gene expression occurs in liver and intestinal cells (3). Numerous population studies have indicated that elevated
LDL cholesterol and apoB protein levels are associated with increased risk of coronary artery disease (1, 14, 15, 22). In contrast, moderately decreased LDL cholesterol levels (6a) are associated with longevity, indicating that the regulation of plasma apoB protein levels is very important. The importance of certain 5' upstream sequences for tissue-specific transcription of the promoterless chloramphenicol acetyltransferase (CAT) gene has been reported (2, 4). One of the studies (4) indicated that apoB gene expresAPOB PROMOTER MUTANTS
-267
CAT ACTIVITY Caco-2 HepG2
(%)
Hela
+8
-267/11
109
108
1.8
87
100
_
19
33
_
)
23
20
-
(-80/-84)
92
40
_
LMI
mm
c-I
10/-1 15)
LM2
Vol. 10, No. 6
0270-7306/90/062653-07$02.00/0 Copyright C) 1990, American Society for Microbiology
Characterization of the Promoter Elements Required for Hepatic and Intestinal Transcription of the Human apoB Gene: Definition of the DNA-Binding Site of a Tissue-Specific Transcriptional Factor DIMITRIS KARDASSIS,1 MARGARITA HADZOPOULOU-CLADARAS,1 DIPAK P. RAMJI,2 RICARDO CORTESE,2 VASSILIS I. ZANNIS,' AND CHRISTOS CLADARASl*
Section of Molecular Genetics, Cardiovascular Institute, Departments of Medicine and Biochemistry, Housman Medical Research Center, Boston University Medical Center, 80 East Concord Street, Boston, Massachusetts 02118,1 and The European Molecular Biology Laboratories, Meyerhofstrasse 1, 6900 Heidelberg, Federal Republic of Germany2 Received 11 September 1989/Accepted 16 February 1990
The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.
Apolipoprotein B is the sole protein component of lowdensity lipoprotein (LDL), constitutes 23.8% of the LDL particle (7, 12), and is involved in the recognition and catabolism of LDL by the LDL receptor (7, 8). The primary structure of human apoB-100 has been determined by derivation of cDNA and genomic and protein sequences (for a review, see reference 27). In humans, apoB gene expression occurs in liver and intestinal cells (3). Numerous population studies have indicated that elevated
LDL cholesterol and apoB protein levels are associated with increased risk of coronary artery disease (1, 14, 15, 22). In contrast, moderately decreased LDL cholesterol levels (6a) are associated with longevity, indicating that the regulation of plasma apoB protein levels is very important. The importance of certain 5' upstream sequences for tissue-specific transcription of the promoterless chloramphenicol acetyltransferase (CAT) gene has been reported (2, 4). One of the studies (4) indicated that apoB gene expresAPOB PROMOTER MUTANTS
-267
CAT ACTIVITY Caco-2 HepG2
(%)
Hela
+8
-267/11
109
108
1.8
87
100
_
19
33
_
)
23
20
-
(-80/-84)
92
40
_
LMI
mm
c-I
10/-1 15)
LM2
