B1-X35S is transcribed by Pol III and AHR increases its transcription with exchange of Pol III by Pol II. (A) IVT using extracts of Hepa-1 cells transiently transfected with empty vector, AHR, or SLUG. Wild-type B1-X35S and mutants B1-Xmut35S and B1-X35Smut were used as templates. (B) B1-X35S expression analyzed by qRT–PCR using total RNA from Hepa-1 cells after transfection with empty vector or AHR. Concentrations of 20 μg/mL α-amanitin (α-AMA) or 5 μg/mL actinomycin-D were added where indicated. (C) The effect of AHR on Pol II binding to wild-type B1-X35S in Hepa-1 cells was analyzed by qChIP. (D) IVT of B1-X35S with extracts of Hepa-1 cells transfected with empty vector or AHR. A total of 10 μM tagetitoxin (TGT) or 3 μg/mL α-AMA were included where indicated. Percentages indicate the inhibition caused by each chemical relative to transcription in corresponding vehicle-treated reactions. (E) IVT as indicated in D using the mutant B1-Xmut35S. (F) IVT using wild-type B1-X35S and extracts of immortalized fibroblasts from AHR-null mice (T-FGM AhR−/−). (C) Average data from the five B1-X35S containing genes Dad1, Lpp, Cabin1, Tbc1d1, and Rtl1. Data are shown as mean ±SD.
