Genes whose expression is highly induced by estradiol (E(2)) contain multiple estrogen response elements (EREs) in their promoters. Previously we reported that estrogen receptor-alpha (ERalpha) binds cooperatively to and E(2) synergistically activates reporter gene expression from three or four tandem copies of a consensus ERE (EREc38). Here we evaluated how ERalpha binding to one, two, three or four tandem copies of EREc38 affects ERalpha conformation as detected by altered ERalpha trypsin digestion patterns in Western blots. E(2)- or 4hydroxytamoxifen (4-OHT)-occupied ERalpha bound to the pS2 ERE or to a single copy of EREc38 showed enhanced susceptibility to trypsin digestion compared to E(2)- or 4-OHT-ERalpha incubated with DNA lacking an ERE. ERalpha binding to multiple tandem copies of EREc38 further increased sensitivity to trypsin digestion. These results correlate with synergistic transcription and cooperativity of ERalpha binding to multiple tandem copies of EREc38. These observations suggest that EREc38 binding alters the overall conformation of ERalpha and that multiple tandem copies of EREc38 enhance these conformational changes. We hypothesize that ERE-induced alterations in ERalpha conformation modulate interaction with coregulatory proteins, resulting in synergistic transcriptional activation.

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