Analysis of estrogen receptor alpha-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 ERalpha and Sp1 in MCF-7 breast cancer cells using fluorescence resonance energy transfer (FRET). Chimeric ERalpha 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 17beta-estradiol, 4'-hydroxytamoxifen, or ICI 182,780. Induction of FRET by these ERalpha 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 ERalpha and Sp1DeltaDBD [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 ERalpha. Results of the FRET assay are consistent with in vitro studies on ERalpha/Sp1 interactions and transactivation, and confirm that ERalpha and Sp1 interact in living breast cancer cells.
Kim
Kyounghyun
K
Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, Veterinary Research Building 409, College Station, Texas 77843-4466, USA.
Barhoumi
Rola
R
Burghardt
Robert
R
Safe
Stephen
S
D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism*; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D047468:Immunoprecipitation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
C485182:Cyan Fluorescent Protein; D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D049452:Green Fluorescent Proteins