Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions.
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.
Wang
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W
Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station 77843-4466, USA.
Dong
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L
Saville
B
B
Safe
S
S
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D004958:Estradiol / Q000494:pharmacology*; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D011401:Promoter Regions, Genetic; D011960:Receptors, Estrogen / Q000378:metabolism* / Q000502:physiology; D056465:Retinoblastoma-Binding Protein 1; D016329:Sp1 Transcription Factor / Q000378:metabolism* / Q000494:pharmacology; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D011960:Receptors, Estrogen; D056465:Retinoblastoma-Binding Protein 1; D016329:Sp1 Transcription Factor; D050680:Transcription Factor DP1; D014157:Transcription Factors; D004958:Estradiol; D004247:DNA