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 modul ate several endocrine systems by altering hormone synthesis, enhancing ligand metabolism, and down-regulating receptor levels/binding activi ty. Previous studies have demonstrated that TCDD inhibits the 17 beta- estradiol (E2)-induction of pS2, a human breast cancer prognostic mark er. This inhibition occurs at the gene expression level and is Ah rece ptor (AhR)-mediated. Analysis of the 5' regulatory region has identifi ed three motifs which resemble dioxin response element (DRE) core sequ ences. pS2-regulated luciferase deletion constructs identified the DRE -like motif located at -527 to -514 as being required for TCDD-mediate d suppression. A point mutation within this core motif (T-518C) abolis hed the inhibition by TCDD while UV-induced protein-DNA cross-linking and competitive gel retardation assays demonstrated AhR complex bindin g to this motif. Further study of this region also revealed an adjacen t putative AP-I site, diverging by one base pair from the consensus se quence. Gel retardation assays using TPA-treated MCF-7 cell nuclear ex tracts showed an induced complex binding to the AP-l-like site. Compet ition studies and antibody supershifts confirmed that the retarded com plex consists of AP-l-like proteins. pS2-regulated luciferase construc ts containing mutations specific to the AP-l-like motif greatly dimini shed the inducibility in response to E2. These results suggest that an interaction between AhR complexes and AP-l-like proteins may be respo nsible for TCDD-mediated inhibition of E2-induced pS2 expression.