FOOTPRINT ANALYSIS OF ESTROGEN-RECEPTOR BINDING TO ADJACENT ESTROGEN RESPONSE ELEMENTS

Quantitative DNase I footprinting assays were employed to simultaneous ly measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). Thes e assays revealed identical, high affinity ER-ERE binding, K-d of appr oximately 0.25 nM, for estradiol-liganded ER (E(2)-ER), 4-hydroxytamox ifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up t o four tandem EREs. Increasing concentrations of ER resulted in the sa me pattern of occupancy for each ERE, whether or not the site was loca ted near other EREs. Similarly, the presence or absence of E(2), 4-OHT , or TAz ligand did not change ER-ERE interaction. Since activated ER- ERE binding affinity is identical, whether ER is liganded or unligande d, ligand cannot regulate ER-ERE binding affinity. These results suppo rt the hypothesis that ligand-dependent conformational changes primari ly determine how ER interacts with components of the transcription ini tiation complex that mediate gene transactivation. In addition, footpr int assays revealed that, following ER binding, an AT-rich site adjace nt to the ERE becomes hypersensitive to DNase I digestion. This sequen ce may be easily or intrinsically bent, assisting in recruiting ER to ERE sites. Copyright (C) 1996 Elsevier Science Ltd.