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 apoAl. To explore th e 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. Electrophoretic mob ility shift assays (EMSA) and Scatchard plot analysis demonstrated tha t yeast produced HNF-4 binds to site A with an affinity two times high er than that of yeast produced RXR alpha. Mapping analysis indicated t hat the 5' portion of site A containing two imperfect direct repeats ( TGAACCCTTGACC) and the sequence of the trinucleotide spacer (CCT) betw een these imperfect repeats are critical determinants for selective bi nding and transactivation by HNF-4. Similar observations were obtained when these mutated versions of site A were evaluated by transient cot ransfection assays in CV1 cells. We conclude that the unique structura l 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.