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. 2013 Sep;41(17):8094-106.
doi: 10.1093/nar/gkt595. Epub 2013 Jul 4.

Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

Maud Marques  1 , Liette Laflamme, Luc Gaudreau
Affiliations

Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

Maud Marques et al. Nucleic Acids Res. 2013 Sep.

Abstract

Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells.

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Figures

Figure 1.
Figure 1.
Estrogen specifically represses CYP1A1 expression in MCF7 cells. CYP1A1 (A) and CYP1B1 (B) mRNA levels were quantified in MCF7 cells grown in estrogen-free media for 3 days, and then treated with DMSO, 10 nM TCDD, 10 nM TCDD + 100 nM E2 or 10 nM TCDD + 500 nM TAM for 24 h. The results are expressed as a percentage of induction in the TCDD-treated sample.
Figure 2.
Figure 2.
H2A.Z depletion impairs AhR-mediated activation in ERα-positive cell lines. (A) MCF7 cells were infected with shCT or shH2A.Z constructs for 5 days, then protein extraction and western blots were performed. Analysis of CYP1A1 and CYP1B1 mRNA expression was performed in ERα-positive cell lines (B) or in ERα-negative cell lines (C). The different cell lines were infected with shCT or shH2A.Z constructs for 5 days and then treated with 10 nM TCDD for 24 h.
Figure 3.
Figure 3.
AhR binding at the CYP1A1 promoter is impaired in H2A.Z-depleted cells or in the presence of E2. (A) Schematic representation of the CYP1A1 promoter. The position of the amplicons A, B, C and D used in the qPCR analyses are illustrated. (B) ChIP of H2A.Z were performed in MCF7 cells treated or not with 10 nM TCDD during 90 min. ChIPs of AhR (C) and RNA polymerase II (D) were performed in MCF7 cells infected with shCT or shH2A.Z constructs for 5 days and then treated or not with 10 nM TCDD during 90 min. ChIPs of AhR (E), ERα (F) and RNA polymerase II (G) were performed in MCF7 cells grown in estrogen-free medium for 3 days, then treated with DMSO, 10 nM TCDD or 10 nM TCDD + 100 nM E2 for 90 min.
Figure 4.
Figure 4.
Inhibition of DNA methylation restores full induction of CYP1A1 and AhR binding at the CYP1A1 promoter in presence of E2. CYP1A1 (A) and CYP1B1 (B) mRNA levels were quantified in MCF7 cells treated with 10 µM 5-azacytidine for 5 days and grown in estrogen-free medium for 3 days, then treated with 10 nM TCDD or 10 nM TCDD + 100 nM E2 during 24 h. ChIPs of AhR (C) and ERα (D) were performed in MCF7 cells treated with 10 µM 5-azacytidine for 5 days and grown in estrogen-free medium for 3 days, then treated with 10 nM TCDD or 10 nM TCDD + 100 nM E2 for 90 min. Primer B was used for the qPCR analysis.
Figure 5.
Figure 5.
ERα can not repress CYP1A1 induction in Dnmt3B-depleted cells. CYP1A1 (A) and CYP1B1 (B) expression was measured in MCF7 cells infected with shCT, shDnmt1, shDnmt3A or shDnmt3B constructs for 5 days and then treated with DMSO (D), 10 nM TCDD (T) or 10 nM TCDD + 100 nM E2 (T+E2) for 24 h. (C) MCF7 cells were infected with shCT, shDnmt1 and shDnmt3b constructs for 5 days, and then proteins were extracted and western blot performed to verify ERα protein levels. Actin is used as loading control. ChIP of Dnmt3B (D) was performed in MCF7 cells grown in estrogen-free media for 3 days, and then treated with DMSO, TCDD or TCDD + E2 for 90 min.
Figure 6.
Figure 6.
ERα induces DNA methylation at the XRE-3 of the CYP1A1 promoter. (A) Schematic representation of the CYP1A1 promoter and XRE positions. Bisulfite sequencing was performed in MCF7 cells infected with shCT or shDnmt3B constructs, grown in estrogen-free media for 3 days and treated with DMSO, TCDD or TCDD + E2 for 24 h. XRE’s are numbered relatively to their positions from the TSS of CYP1A1, and each red rectangles represent one XRE. (B) Graphical representation of the percentage of unmethylated and methylated CpGs in XRE-3 (*P < 0.05).
Figure 7.
Figure 7.
H2A.Z depletion promotes DNA methylation at the CYP1A1 promoter. (A) Schematic representation of the CYP1A1 promoter. The position of the amplicons A and B used in the qPCR analyses are illustrated. (B) MeDIP was performed in MCF7 cells infected with shCT or shH2A.Z constructs for 5 days. (C) Bisulfite sequencing were performed in MCF7 cells infected with shCT or shH2A.Z constructs and grown in DMEM 10% FBS for 5 days.
Figure 8.
Figure 8.
Proposed model for CYP1A1 gene regulation by AhR and ERα. (A) In the absence of estradiol, when TCDD is added in the media, AhR /Arnt binds the XRE’s located in the CYP1A1 promoter. At the same time, the histone variant H2A.Z is removed from the XRE’s. (B) In the presence of estradiol, ERα displaces AhR /Arnt by promoting DNA methylation on the XRE’s of the CYP1A1 promoter, thus resulting in less AhR activating surfaces available to stimulate CYP1A1 expression.
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