Analysis of estrogen receptor α-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer

Estrogen-dependent regulation of several genes associated with cell cycle progression, proliferation, and nucleotide metabolism in breast cancer cells is associated with interactions of estrogen receptor (ER)alpha/Sp1 with GC-rich promoter elements. This study investigates ligand-dependent interactions of ER alpha and Sp1 in MCF-7 breast cancer cells using fluorescence resonance energy transfer (FRET). Chimeric ER alpha and Sp1 proteins fused to cyan fluorescent protein or yellow fluorescent protein were transfected into MCF-7 cells, and a FRET signal was induced after treatment with 17 beta-estradiol, 4'-hydroxytamoxifen, or ICI 182,780. Induction of FRET by these ER alpha agonists/antagonists was paralleled by their activation of gene expression in cells transfected with a construct (pSp1(3)) containing three tandem Sp1 binding sites linked to a luciferase reporter gene. In contrast, interactions between ER alpha and Sp1 Delta DBD [a DNA binding domain (DBD) deletion mutant of Sp1] are not observed, and this is consistent with the critical role of the C-terminal DBD of Sp1 for interaction with ER alpha. Results of the FRET assay are consistent with in vitro studies on ER alpha/Sp1 interactions and transactivation, and confirm that ER alpha and Sp1 interact in living breast cancer cells.