Ah receptor regulation of mouse Cyp1B1 is additionally modulated by a second novel complex that forms at two AhR response elements.

ABSTRACT

Cyp1B1 is expressed constitutively in many extrahepatic cells and is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). An enhancer region (AhER-810 to -1075 of the mouse Cyp1B1 promoter), which mediates aryl hydrocarbon receptor (AhR) regulation of transcription, contains three consensus XRE sequences (designated XRE1, XRE4, and XRE5) and a central Ebox. XRE5 is essential for both basal and induced activity in C3H10T1/2 cells. AhR/ARNT binding to XRE1, XRE4, and the Ebox complex function in combination to support the AhR/ARNT complex at XRE5. The identical 12 base cores of XRE1 and XRE4 differ from the core of XRE5 by two bases outside of the consensus XRE. These sites bind a constitutive complex slightly smaller than AhR/ARNT (anomalous complex; anC), which is not formed at XRE5 or six Cyp1A1 XREs. Exchange of these bases (m3 mutations) restores selective AhR/ARNT binding at XRE1/XRE4 and introduces anC binding at XRE5. The activities of multimeric XRE1 and XRE5 luciferase reporters responded in parallel to the extent of AhR/ARNT binding. The consensus anC binding sequence ((C/T)GCG(C/T)GCGC(C/A)GC) overlaps the XRE1/XRE4 AhR/ARNT element. Gel mobility analyses show that anC binds to XRE1/XRE4 under basal conditions, while AhR/ARNT partially displaces anC following TCDD induction. Selective depletion of anC with biotin-oligonucleotides increases AhR/ARNT binding. M3-mutations at, respectively, XRE1 and XRE4 of the AhER sequence, had opposite effects on luciferase reporters. Activities increased for the XRE1 mutation and decreased for the XRE4 mutation, but also depended on the level of AhR transfected into AhR -/- fibroblasts. AnC compete with AhR at XRE1 while playing an activating role at XRE4. This positive effect of constitutive anC binding at XRE4 may contribute to the characteristic basal Cyp1B1 expression in embryo fibroblasts, which is mediated by low constitutive activities of AhR.