Interaction of a liver-specific factor with an enhancer 4.8 kilobases upstream of the phosphoenolpyruvate carboxykinase gene.
We have previously identified a series of five DNase-I hypersensitive (HS) sites within and around the rat phosphoenolpyruvate carboxykinase (PEPCK) gene. The far upstream region has now been sequenced, and the tissue-specific HS site has been mapped more precisely at 4,800 base pairs upstream of the transcription start site of the PEPCK gene. DNA fragments that include the HS site were cloned upstream of various promoters to test whether these regions modulate transcription of the chloramphenicol acetyltransferase reporter gene. Chloramphenicol acetyltransferase activity was enhanced when the DNA fragment encompassing the upstream HS site was linked to various lengths of the PEPCK promoter or to the heterologous simian virus 40 promoter. This upstream region in conjunction with the proximal promoter, which may contain a tissue-specific element, conferred maximum activation in H4IIE hepatoma cells, which express the endogenous PEPCK gene. When these experiments were performed in XC cells, in which the gene is not expressed, transcriptional activation by the upstream element was still significant. Evidence of a specific protein-DNA interaction, using DNA mobility shift and DNase I footprinting assays, was obtained only when using H4IIE cell nuclear extracts. Competition assay showed that the interacting factor may be similar or identical to the liver-specific factor HNF3. We suggest that this protein factor binds to DNA within the HS site and interacts with the proximal promoter region to control tissue-specific high-level expression of the PEPCK gene.
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Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Poon
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Stone
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Granner
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Chalkley
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002843:Chromatin / Q000502:physiology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D005796:Genes; D008099:Liver / Q000201:enzymology; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic
D002843:Chromatin; D004268:DNA-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I; D010729:Phosphoenolpyruvate Carboxykinase (GTP)