Fig. 7. Agonist-specific AhR target-gene expression transfers with the gene-specific “XRE cassette” in the context of chromatin architecture. An edited AML12 cell line was constructed by replacing the 926-bp cyp1a1 promoter region containing 10 XREs (between −574 and −1500 bp from the transcription start site) with the stc2 promoter containing eight XREs (the 259-bp region termed “stc2 XRE cassette” – between −210 and −469 bp from the transcription start site) using CRISPR/Cas9 technology. (A) Illustration of cyp1a1 and stc2 promoter regions. Red and blue rectangles represent XREs (5′-GCGTG-3′) within cyp1a1 and stc2 promoters respectively. CRISPR/Cas9-edited AML12 cells, wherein the 259-bp stc2 XRE cassette is inserted by replacing cyp1a1 XREs within −574 and −1500 bp, is depicted. (B) WT (black bars) and CRISPR/Cas9-edited (gray bars) AML12 cells were treated with vehicle (DMSO), 6 nM TCDD, and 30 µM CA for 2 hours. Quantitative RT-PCR was performed to measure RNA expression of cyp1a1 and stc2 and normalized to 18S rRNA. For statistical analysis, a mixed-effects multivariate ANOVA (MANOVA) model was used. After overall significant F test from MANOVA model, the post hoc multiple-comparison tests were performed by Tukey procedure. *P < 0.05, n = 3 independent batches of AML12 cells. (C) Vehicle-, TCDD-, and CA-treated, WT and edited AML12 cells were subjected to chromatin immunoprecipitation using antibodies against AhR, H4 K5ac, Atf2, H3 K79me, Dot1l, and H3 (positive control). PCR products were fractionated and visualized on 5% polyacrylamide gels stained with SYBR Green. Samples were run on separate gels (represented by space), stained with SYBR Green, and imaged on Chemidoc MP imager (Bio-Rad) synchronously with exactly the same acquisition parameters (n = 3 independent batches of AML12 cells).