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Nucleic Acids Research, 1994, Vol. 22, No. 25 5665-5671
© 1994

Articles

Transcriptional regulation of the apoAl gene by hepatic nuclear factor 4 in yeast

Hans A. Fuernkranz, Yisheng Wang, Sotirios K. Karathanasis and Paul Mak*

Department of Cardiovascular Molecular Biology, Lederle Laboratories 401 N. Middletown Road, American Cyanamid Co., Pearl River, NY 10965, USA

*To whom correspondence should be addressed

Received August 23, 1994. Revised October 24, 1994. Accepted October 24, 1994.

Hepatocyte Nuclear Factor 4 (HNF-4), a liver-enriched orphan receptor of the nuclear receptor superfamlly, is required for the expression of a wide variety of liver-specific genes including apoAl. To explore the possibility that site A of the apoAl gene enhancer might also be the target for HNF-4 without the interference of endogenous mamalian cell proteins that also bind to site A, we tested the ability of HNF-4 to activate transcription from site A in yeast cells. Electrophoretlc 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 RXRa. 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 transactlvation 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 apoAl gene regulation.


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