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. 2016 Oct 7:6:34989.
doi: 10.1038/srep34989.

Dioxin induces Ahr-dependent robust DNA demethylation of the Cyp1a1 promoter via Tdg in the mouse liver

Hesbon Z Amenya  1 , Chiharu Tohyama  1   2 , Seiichiroh Ohsako  1
Affiliations

Dioxin induces Ahr-dependent robust DNA demethylation of the Cyp1a1 promoter via Tdg in the mouse liver

Hesbon Z Amenya et al. Sci Rep. .

Abstract

The aryl hydrocarbon receptor (Ahr) is a highly conserved nuclear receptor that plays an important role in the manifestation of toxicity induced by polycyclic aromatic hydrocarbons. As a xenobiotic sensor, Ahr is involved in chemical biotransformation through activation of drug metabolizing enzymes. The activated Ahr cooperates with coactivator complexes to induce epigenetic modifications at target genes. Thus, it is conceivable that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent Ahr ligand, may elicit robust epigenetic changes in vivo at the Ahr target gene cytochrome P450 1a1 (Cyp1a1). A single dose of TCDD administered to adult mice induced Ahr-dependent CpG hypomethylation, changes in histone modifications, and thymine DNA glycosylase (Tdg) recruitment at the Cyp1a1 promoter in the liver within 24 hrs. These epigenetic changes persisted until 40 days post-TCDD treatment and there was Cyp1a1 mRNA hyperinduction upon repeat administration of TCDD at this time-point. Our demethylation assay using siRNA knockdown and an in vitro methylated plasmid showed that Ahr, Tdg, and the ten-eleven translocation methyldioxygenases Tet2 and Tet3 are required for the TCDD-induced DNA demethylation. These results provide novel evidence of Ahr-driven active DNA demethylation and epigenetic memory. The epigenetic alterations influence response to subsequent chemical exposure and imply an adaptive mechanism to xenobiotic stress.

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Figures

Figure 1
Figure 1. A single dose of TCDD induces Cyp1a1 transcriptional memory.
C57BL/6J female mice (wild type) were orally treated with TCDD at a single dose of 3 μg/kg bw, and livers were collected at indicated time points post-TCDD exposure. (a) Time-course changes of Cyp1a1 mRNA expression levels in TCDD-treated animals (n = 3). (b) Lack of response in Ahr−/−animals. (c) Wild type female animals were pretreated with TCDD and repeat treated 40 days after the initial dose. Note an approximately three-fold super-induction of Cyp1a1 in TCDD pretreated animals in comparison with non-pretreated animals (n = 3). *P < 0.05, Student’s t-test.
Figure 2
Figure 2. ChIP assay for histone modifications at the Cyp1a1 promoter.
C57BL/6J mice were treated with 3 μg/kg of TCDD and livers were collected at 24 hrs and 40 days post-TCDD treatment. (a–c) Levels of the histone modifications H3K4me3, H4ac and H4K20me3 as assayed by ChIP assay, 24 hrs after TCDD exposure, at the promoter regions indicated. (d–f) The histone modifications H3K4me3, H4ac, and H4K20me3 as assayed by ChIP assay, 40 days after TCDD exposure. Data are expressed as mean ± SE. (n = 3). *P < 0.05, Student’s t-test.
Figure 3
Figure 3. TCDD induces Ahr-dependent CpG demethylation of the Cyp1a1 promoter.
Animals were treated with 3 μg/kg TCDD and livers were sampled at the indicated time points. (a,b) The methylation level of the −500 CpG and −420 CpG, respectively, in the Cyp1a1 promoter as measured by MSRE-qPCR. Data are expressed as mean ± SE. (n = 3). *P < 0.05, two-way ANOVA followed by Fisher's PLSD post hoc test.
Figure 4
Figure 4. Ahr-driven active demethylation involves 5 hmC and active DNA demethylation mediators Apex1, Tdg, and Tet3.
(a) 5-hydroxymethylcytosine levels after TCDD exposure. 5 hmC was analyzed by 5 hmC sensitive restriction enzyme digestion and qPCR. Data are expressed as mean ± SE. (n = 3). *P < 0.05, two-way ANOVA followed by Fisher’s PLSD post hoc test. Cyp1a1 promoter occupancy of demethylation mediators as analyzed by ChIP assay in adult mouse livers; (b) Apex 1, (c) Tdg, (d) Tet3. Data are expressed as mean ± SE. (n = 3). *P < 0.05, Student’s t-test.
Figure 5
Figure 5. Ahr, Tdg, Tet2, and Tet3 are required for Ahr-directed DNA demethylation.
(a) Hepa1c1c7 endogenous Cyp1a1 promoter methylation levels at the target CpGs as analyzed by MSRE-qPCR. (b) The in vitro methylated Cyp1a1 reporter plasmid response to TCDD as analyzed by luciferase expression. 100 ng of the methylated plasmid was transfected into Hepa1c1c7 cells. After treatment with 10 nM TCDD for 24 hrs, luciferase activity was measured by the Dual-Luciferase® Reporter Assay System. (c,d) methylation levels of the −500 and −420 CpG sites respectively, in the in vitro methylated plasmids after TCDD exposure. Data are expressed as mean ± SE. (n = 3). *P < 0.05, Student’s t-test. (e) siRNA knockdown efficiency 48 hrs after transfection; mRNA levels were measured by RT-qPCR and protein levels by western blot. (n = 2). (f) Methylation level of the methylated plasmid after siRNA target knockdown and TCDD treatment. Data are expressed as mean ± SE. (n = 3). *P < 0.05, one-way ANOVA followed by Fisher's PLSD post hoc test.
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