Identification of a motif within the 5' regulatory region of pS2 which is responsible for AP-1 binding and TCDD-mediated suppression.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds modulate several endocrine systems by altering hormone synthesis, enhancing ligand metabolism, and down-regulating receptor levels/binding activity. Previous studies have demonstrated that TCDD inhibits the 17beta-estradiol (E2)-induction of pS2, a human breast cancer prognostic marker. This inhibition occurs at the gene expression level and is Ah receptor (AhR)-mediated. Analysis of the 5' regulatory region has identified three motifs which resemble dioxin response element (DRE) core sequences. pS2-regulated luciferase deletion constructs identified the DRE-like motif located at -527 to -514 as being required for TCDD-mediated suppression. A point mutation within this core motif (T-518C) abolished the inhibition by TCDD while UV-induced protein-DNA cross-linking and competitive gel retardation assays demonstrated AhR complex binding to this motif. Further study of this region also revealed an adjacent putative AP-1 site, diverging by one base pair from the consensus sequence. Gel retardation assays using TPA-treated MCF-7 cell nuclear extracts showed an induced complex binding to the AP-1-like site. Competition studies and antibody supershifts confirmed that the retarded complex consists of AP-1-like proteins. pS2-regulated luciferase constructs containing mutations specific to the AP-1-like motif greatly diminished the inducibility in response to E2. These results suggest that an interaction between AhR complexes and AP-1-like proteins may be responsible for TCDD-mediated inhibition of E2-induced pS2 expression.
Gillesby
B E
BE
Department of Pharmacology and Toxicology, University of Western Ontario, London, Canada.
Stanostefano
M
M
Porter
W
W
Safe
S
S
Wu
Z F
ZF
Zacharewski
T R
TR
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011485:Protein Binding; D011506:Proteins / Q000235:genetics*; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D000071163:Trefoil Factor-1; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D011506:Proteins; D018336:Receptors, Aryl Hydrocarbon; D011960:Receptors, Estrogen; C051838:TFF1 protein, human; D018808:Transcription Factor AP-1; D000071163:Trefoil Factor-1; D025521:Tumor Suppressor Proteins; D004958:Estradiol; D013755:Tetradecanoylphorbol Acetate