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

Mol Endocrinol. 2005 Apr;19(4):843-54. doi: 10.1210/me.2004-0326. Epub 2005 Jan 6.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Breast Neoplasms / metabolism*
  • Cell Nucleus / chemistry
  • Cytoplasm / chemistry
  • DNA-Binding Proteins / analysis
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Estrogen Receptor alpha / analysis
  • Estrogen Receptor alpha / genetics
  • Estrogen Receptor alpha / metabolism*
  • Female
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins / analysis
  • Humans
  • Immunoprecipitation
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Sp1 Transcription Factor / analysis
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / metabolism*

Substances

  • Cyan Fluorescent Protein
  • DNA-Binding Proteins
  • Estrogen Receptor alpha
  • Recombinant Fusion Proteins
  • Sp1 Transcription Factor
  • Green Fluorescent Proteins