Identification of novel steroid-response elements

Abstract

A rapid method for defining novel steroid-responsive elements has been developed. Large libraries of degenerate oligonucleotides were analyzed using a yeast-based screen to identify estrogen-responsive DNA sequences. From a library of 40,000 recombinants, seven estrogen-responsive clones were identified. When sequenced, these elements showed remarkable diversity and were different from the consensus vitellogenin A2 ERE. One surprising result was the presence of the two half sites as direct repeats in some of the clones. This implies that in vivo estrogen receptor can bind and transactivate yeast genes through response elements in which the two half sites align as direct repeats. This protocol requires no purified protein and specifically selects for functional response elements. It has a wide application in the study of any transcription factor/DNA interaction.

Figure 1

Western immunoblot analysis of hER expressed in yeast. Yeast extract (50 μg of protein) from YEPE10 was analyzed by immunoblot as described in Materials and Methods, using D-75 ER antibody.

Figure 2

Identification of estrogen response elements using a yeast-based screen. A library of degenerate oligonucleotides YRP(SRE)n was created by cloning the double-stranded oligonucleotides into the unique Bgl II site of yeast reporter plasmid YRPC3. CYC1 is the yeast iso-1-cytochrome C promoter fused to the structural gene of E. coli Lac Z. URA3 is the selective marker for uracil. 2 Micron is the replicating DNA of yeast. The library was introduced into yeast cells carrying the estrogen receptor expression plasmid (YEPE10), and the cells were selected for tryptophan and uracil auxotrophy on plates containing X-gal and 17β-estradiol. Transformants developing blue color were picked, and functional estrogen response elements were selected.

Figure 3

Transcriptional activity of hER from reporter plasmids containing receptor binding sites as direct or indirect repeats. The yeast cells containing YEPE10 and YRPE2 plasmids and the cells containing YEPE10 and YRPD2 plasmids were grown in minimal media in the absence and presence of estradiol with increasing concentrations of copper sulfate (CuS04). Cytosols were prepared, and induction of lacZ was measured. The β-galactosidase activity measured in yeast extracts is expressed as Miller units per mg protein.