Identification of a motif within the 5' regulatory region of pS2 which is responsible for AP-1 binding and TCDD-mediated suppression

Bradley E. Gillesby, Michael Stanostefano, Weston Porter, Stephen Safe, Zhi Fen Wu, Timothy R. Zacharewski

Research output: Contribution to journalArticlepeer-review

126 Scopus citations

Abstract

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 17β-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.

Original languageEnglish (US)
Pages (from-to)6080-6089
Number of pages10
JournalBiochemistry
Volume36
Issue number20
DOIs
StatePublished - May 20 1997

ASJC Scopus subject areas

  • Biochemistry

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