Retinoid X receptor homodimers function as transcriptional activators in yeast.
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.
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Department of Cardiovascular Molecular Biology, Lederle Laboratories, American Cyanamid Co., Pearl River, NY 10965.
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D001483:Base Sequence; D015153:Blotting, Western; D051836:COUP Transcription Factors; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D047488:Retinoid X Receptors; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors
D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D047488:Retinoid X Receptors; D015534:Trans-Activators; D014157:Transcription Factors