Functional analysis of the human estrogen receptor using a phenotypic transactivation assay in yeast.
We have constructed yeast strains in which the expression of the Saccharomyces cerevisiae URA3 gene is induced by the human estrogen receptor (hER). Promoter sequences required for both basal and activated transcription of URA3 were replaced with one or three estrogen-response elements (EREs) positioned upstream of the native TATA box. These constructs were each integrated at the TRP1 locus of a yeast strain in which the natural URA3 gene had been deleted, and the integrants were transformed with low- or high-copy-number shuttle plasmids expressing wild-type or truncated derivatives of hER. Transformants were assayed for growth on uracil-deficient medium plus or minus estradiol (E2), for resistance to 5-fluoroorotic acid (5-FOA) and for activity of OMPdecase (orotidine-5'-monophosphate decarboxylase), the product of the URA3 gene. We show that the growth and 5-FOA-resistance (5-FOAR) phenotypes of these strains are strictly dependent upon the function of the receptor derivatives. Induction of URA3, measured by OMPdecase activity, was observed over a 20- to 2500-fold range depending on the receptor derivative, its expression level and the number of EREs in the responsive promoter. Both one- and three-ERE reporter strains expressing the full-length receptor are completely E2-dependent for growth, and display a 5-FOAR phenotype in the absence of the hormone. We demonstrate that the individual hER transactivation functions, TAF1 and TAF2, are both functional in yeast, and that the hormone-dependent TAF2 is the more potent activator on our reporters. We show that hER displays strong homosynergism in yeast, and discuss the contributions of the two TAFs in hER synergism.(ABSTRACT TRUNCATED AT 250 WORDS)
Pierrat
B
B
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de l'INSERM, Faculté de Médecine, Strasbourg, France.
Heery
D M
DM
Lemoine
Y
Y
Losson
R
R
D015153:Blotting, Western; D003001:Cloning, Molecular; D004958:Estradiol / Q000378:metabolism; D006801:Humans; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics; D010641:Phenotype; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation
D011960:Receptors, Estrogen; D015534:Trans-Activators; D004958:Estradiol; D009964:Orotidine-5'-Phosphate Decarboxylase