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
| Autor: | Meghan Sullivan, Beatriz Levy-Wilson, Travis J. Antes, Stephen G. Young, Cathy Huynh, Sheryl A. Goodart |
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| Rok vydání: | 2000 |
| Předmět: |
Apolipoprotein B
Molecular Sequence Data Restriction Mapping Mice Transgenic Enhancer RNAs Deoxyribonuclease HindIII Biology Biochemistry Deoxyribonuclease EcoRI Mice Ribonucleases Animals Deoxyribonuclease I Humans Transgenes Intestinal Mucosa Enhancer Molecular Biology Transcription factor Apolipoproteins B Base Sequence Basic Helix-Loop-Helix Leucine Zipper Transcription Factors Structural gene Nuclear Proteins Cell Biology Transfection Phosphoproteins Molecular biology DNA-Binding Proteins Hepatocyte nuclear factors Enhancer Elements Genetic Hepatocyte Nuclear Factor 4 Hepatocyte nuclear factor 4 Hepatocyte Nuclear Factor 3-beta biology.protein Electrophoresis Polyacrylamide Gel Sequence Alignment Transcription Factors |
| Zdroj: | Journal of Biological Chemistry. 275:26637-26648 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m003025200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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