Transcriptional regulation of the apoAI gene by hepatic nuclear factor 4 in yeast.
Hepatocyte Nuclear Factor 4 (HNF-4), a liver-enriched orphan receptor of the nuclear receptor superfamily, is required for the expression of a wide variety of liver-specific genes including apoAI. To explore the possibility that site A of the apoAI gene enhancer might also be the target for HNF-4 without the interference of endogenous mammalian cell proteins that also bind to site A, we tested the ability of HNF-4 to activate transcription from site A in yeast cells. Electrophoretic mobility shift assays (EMSA) and Scatchard plot analysis demonstrated that yeast produced HNF-4 binds to site A with an affinity two times higher than that of yeast produced RXR alpha. Mapping analysis indicated that the 5' portion of site A containing two imperfect direct repeats (TGAACCCTTGACC) and the sequence of the trinucleotide spacer (CCT) between these imperfect repeats are critical determinants for selective binding and transactivation by HNF-4. Similar observations were obtained when these mutated versions of site A were evaluated by transient cotransfection assays in CV1 cells. We conclude that the unique structural determinants of site A in conjunction with the differential binding affinity of HNF-4 for site A may play a fundamental role in apoAI gene regulation.
Department of Cardiovascular Molecular Biology, Lederle Laboratories, American Cyanamid Co., Pearl River, NY 10965.
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Fuernkranz
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Wang
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Karathanasis
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D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D051557:Hepatocyte Nuclear Factor 4; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010750:Phosphoproteins; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D047488:Retinoid X Receptors; D012441:Saccharomyces cerevisiae; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection
D016632:Apolipoprotein A-I; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors