Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions

Mol Endocrinol. 1999 Aug;13(8):1373-87. doi: 10.1210/mend.13.8.0323.

Abstract

17beta-Estradiol (E2) stimulated proliferation and DNA synthesis in MCF-7 human breast cancer cells, and this was accompanied by induction of E2F1 mRNA and protein levels. Analysis of the E2F1 gene promoter showed that the -146 to -54 region was required for E2-responsiveness in transient transfection assays, and subsequent deletion/mutation analysis showed that a single upstream GC-rich and two downstream CCAAT-binding sites were required for transactivation by E2. Gel mobility shift assays with multiple oligonucleotides and protein antibodies (for supershifts) showed that the -146 to -54 region of the E2F1 gene promoter bound Sp1 and NF-Y proteins in MCF-7 cells. The estrogen receptor (ER) protein enhanced Sp1 interactions with upstream GC-rich sites, and interactions of ER, Sp1, and ER/Sp1 with downstream DNA bound-NF-Y was investigated by kinetic analysis for protein-DNA binding (on- and off-rates), coimmunoprecipitation, and pulldown assays using wild-type and truncated glutathione S-transferase (GST)-Sp1 chimeric proteins. The results showed that Sp1 protein enhanced the Bmax of NF-Y-DNA binding by more than 5-fold (on-rate); in addition, the Sp1-enhanced NF-Y-DNA complex was further stabilized by coincubation with ER and the rate of dissociation (t1/2) was decreased by approximately 50%. Sp1 antibodies immunoprecipitated [35S]NF-YA after coincubation with unlabeled Sp1 protein. Thus, transcriptional activation of E2F1 gene expression in MCF-7 cells by E2 is regulated by multiprotein ER/Sp1-NF-Y interactions at GC-rich and two CCAAT elements in the proximal region of the E2F1 gene promoter. This represents a unique trans-acting protein complex in which ligand-dependent transactivation by the ER is independent of direct ER interactions with promoter elements.

Publication types

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

MeSH terms

  • Breast Neoplasms / genetics*
  • Breast Neoplasms / metabolism
  • CCAAT-Enhancer-Binding Proteins
  • Carrier Proteins*
  • Cell Cycle Proteins*
  • DNA / metabolism
  • DNA-Binding Proteins / metabolism*
  • DNA-Binding Proteins / pharmacology
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • Estradiol / pharmacology*
  • Gene Deletion
  • Gene Expression / drug effects
  • Humans
  • Promoter Regions, Genetic
  • Receptors, Estrogen / metabolism*
  • Receptors, Estrogen / physiology
  • Retinoblastoma-Binding Protein 1
  • Sp1 Transcription Factor / metabolism*
  • Sp1 Transcription Factor / pharmacology
  • Transcription Factor DP1
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcription, Genetic
  • Transfection
  • Tumor Cells, Cultured

Substances

  • CCAAT-Enhancer-Binding Proteins
  • Carrier Proteins
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • E2F1 protein, human
  • Receptors, Estrogen
  • Retinoblastoma-Binding Protein 1
  • Sp1 Transcription Factor
  • Transcription Factor DP1
  • Transcription Factors
  • Estradiol
  • DNA