Estrogen response element binding induces alterations in estrogen receptor-α conformation as revealed by susceptibility to partial proteolysis

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 (ER alpha) 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 ERa binding to one, two, three or four tandem copies of EREc38 affects ERa conformation as detected by altered ERa trypsin digestion patterns in Western blots. E-2- or 4-hydroxytamoxifen (4-OHT)-occupied ER alpha 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-ER alpha incubated with DNA lacking an ERE. ERa binding to multiple tandem copies of EREc38 further increased sensitivity to trypsin digestion. These results correlate with synergistic transcription and cooperativity of ER alpha binding to multiple tandem copies of EREc38. These observations suggest that EREc38 binding alters the overall conformation of ERa and that multiple tandem copies of EREc38 enhance these conformational changes. We hypothesize that ERE-induced alterations in ERa conformation modulate interaction with coregulatory proteins, resulting in synergistic transcriptional activation.