ABSTRACT
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.
Subject(s)
Apolipoprotein A-I/genetics , Phosphoproteins , Transcription Factors/physiology , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Cell Line , Chlorocebus aethiops , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation , Hepatocyte Nuclear Factor 4 , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Saccharomyces cerevisiae , Transcription Factors/metabolism , TransfectionABSTRACT
The possibility that different retinoids activate transcription from a specific retinoic acid (RA)-responsive element known as site A via different homo and heterodimeric versions of RA receptors cannot be evaluated in mammalian cells because they contain endogenous RA receptors (RAR). However, this limitation can be overcome by using yeast cells, which do not contain endogenous RAR, to study retinoid signaling pathways. Here, we describe heterologous expression of the human retinoid X receptor (RXR alpha) in yeast and hormone-dependent activation of a reporter construct containing site A upstream from a yeast promoter fused to the lacZ gene of Escherichia coli. Western blot analysis of yeast extracts containing RXR alpha revealed a distinct immunoreactive polypeptide co-migrating with the mammalian-produced RXR alpha. Electrophoretic mobility shift assays demonstrated that RXR alpha produced in yeast binds efficiently to site A in the absence of 9-cis-RA. However, transcription activation experiments showed that RXR alpha transactivates a yeast basal promoter linked to site A only in the presence of 9-cis-RA. We conclude that RXR alpha homodimers bind to site A in the absence of 9-cis-RA, but function as ligand-dependent transactivators in yeast cells. This retinoid-responsive transcription unit created in yeast cells provides a powerful genetic tool for the systemic unraveling of the synergistic interactions between RXR alpha and its heterodimeric partners.
Subject(s)
Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid , Receptors, Steroid , Saccharomyces cerevisiae/metabolism , Trans-Activators/metabolism , Transcription Factors , Base Sequence , Blotting, Western , COUP Transcription Factors , Cloning, Molecular , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Promoter Regions, Genetic , Retinoid X Receptors , Saccharomyces cerevisiae/genetics , Signal TransductionABSTRACT
The tissue distributions of the estrogen-inducible hen oviduct transferrin receptor and the chicken embryonic erythrocyte transferrin receptor were studied. Tissue sections were investigated by immunofluorescence microscopy using specific polyclonal antisera against each receptor. The receptor originally identified and characterized in the oviduct strongly stained liver and ovary; localized staining was observed in the brain (especially endothelial cells). Staining of breast muscle and heart tissue occurred only in occasional interstitial cells. Antiserum against oviduct transferrin receptor did not stain erythrocytes, either from embryos or from mature animals. On the other hand, the antiserum directed against the embryonic erythrocyte receptor stained embryonic erythrocytes; it did not stain any other tissues. The fluorescence microscopy observations were confirmed by Western blot analysis. The strong staining of oviduct, liver, and ovary suggests a major role for the oviduct transferrin receptor in oogenesis.
