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-specific regulatory elements that control apoB expression in the intestines of transgenic mice. In this study, we further localized the apoB intestinal control region to a 3-kb segment (-54 to -57 kb). DNaseI hypersensitivity studies uncovered a prominent DNaseI hypersensitivity site, located within a 315-base pair (bp) fragment at the 5'-end of the 3-kb segment, in transcriptionally active CaCo-2 cells but not in transcriptionally inactive HeLa cells. Transient transfection experiments with CaCo-2 and HepG2 cells indicated that the 315-bp fragment contained an intestine-specific enhancer, and analysis of the DNA sequence revealed putative binding sites for the tissue-specific transcription factors hepatocyte nuclear factor 3beta, hepatocyte nuclear factor 4, and CAAT enhancer-binding protein beta. Binding of these factors to the 315-bp enhancer was demonstrated in gel retardation experiments. Transfection of deletion mutants of the 315-bp enhancer revealed the relative 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 of sequence conservation in the binding sites for the key transcriptional activators and also exhibited enhancer activity in transient transfection assays with CaCo-2 cells. In transgenic mouse expression studies, the 315-bp enhancer conferred intestinal expression to human apoB transgenes.
Antes
T J
TJ
Research Institute, Palo Alto Medical Foundation, Palo Alto, California 94301, USA.
Goodart
S A
SA
Huynh
C
C
Sullivan
M
M
Young
S G
SG
Levy-Wilson
B
B
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics*; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D051542:Hepatocyte Nuclear Factor 3-beta; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D015183:Restriction Mapping; D012260:Ribonucleases / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000378:metabolism; D019076:Transgenes
D001055:Apolipoproteins B; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C496503:Foxa2 protein, mouse; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; C501970:Tcfl4 protein, mouse; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D012260:Ribonucleases; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D003850:Deoxyribonuclease I