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

Kyounghyun Kim, Rola Barhoumi, Robert Burghardt, Stephen Safe

Research output: Contribution to journalArticle

54 Scopus citations

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)α/Sp1 with GC-rich promoter elements. This study investigates ligand-dependent interactions of ERα and Sp1 in MCF-7 breast cancer cells using fluorescence resonance energy transfer (FRET). Chimeric ERα 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 170-estradiol, 4′-hydroxyitamoxifen, or ICI 182,780. Induction of FRET by these ERα agonists/antagonists was paralleled by their activation of gene expression in cells transfected with a construct (pSp13) containing three tandem Sp1 binding sites linked to a luciferase reporter gene. In contrast, interactions between ERα and Sp1ΔDBD [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 ERα. Results of the FRET assay are consistent with in vitro studies on ERα/Sp1 interactions and transactivation, and confirm that ERα and Sp1 interact in living breast cancer cells.

Original languageEnglish (US)
Pages (from-to)843-854
Number of pages12
JournalMolecular Endocrinology
Volume19
Issue number4
DOIs
StatePublished - Apr 2005

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

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