Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: A positive selection system

The yeast URA3 gene was used as a reporter to investigate the activities of estrogenic and antiestrogenic compounds in yeast Saccharomyces cerevisiae. The control sequences of the wild type (wt) URA3 promoter were replaced with zero, two, or six copies of estrogen-response elements (ERE). Insertion of two and six co:pies of ERE rendered the expression of the URA3 gene to be dependent on the presence of the human estrogen receptor (ER) and the hormone 17 beta-estradiol (E(2)). Two versions of the ER genes were constructed: a full-length wild-type ER (ERa-f) and a truncated ER with domains C, D, and E (ERcde). Both forms of the ER were able to activate the ERE-URA3 reporter in a hormone-dependent manner. The growth of yeast transformants were hormone-dependent when the reporter constructs were inserted into chromosomes using yeast integrating vectors (YIp) but not with the 2 mu-based episomal (high-copy number, YEp) or centromeric (low-copy number, YCp) vectors. The integrated transformants were employed to investigate the effects of estrogenic and antiestrogenic compounds. The estrogenic compounds, E(2), diethylstilbestrol (DES), and estrone (EST), activated express:ion of the reporter genes at 1 nM concentration, which is the same concentration exhibiting activity in mammalian cells. None of the antiestrogens, at concentrations up to 1 mu M, including tamoxifen (TAM), raloxifene (RAL), and ICI 164,384 (ICI) antagonized 1 nM of E, against either form of the ER. In fact, TAM, RAL, and ICI displayed slight agonistic activity at high concentra;tions of 300 nM. or greater to the ERcde. This system can be used to investigate or clone the missing factor(s) that is responsible for the antagonistic activity of the ER in yeast, and is also suitable for screening for the effecters of the ER.