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. 2010 Jan 5;5(1):e8579.
doi: 10.1371/journal.pone.0008579.

Nrf2 expression is regulated by epigenetic mechanisms in prostate cancer of TRAMP mice

Siwang Yu  1 , Tin Oo Khor, Ka-Lung Cheung, Wenge Li, Tien-Yuan Wu, Ying Huang, Barbara A Foster, Yuet Wai Kan, Ah-Ng Kong
Affiliations

Nrf2 expression is regulated by epigenetic mechanisms in prostate cancer of TRAMP mice

Siwang Yu et al. PLoS One. .

Abstract

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Hypermethylation of Nrf2 promoter in TRAMP prostate was correlated with tumorigenesis.
(A) A CpG island was identified in the 5′-flanking region of mouse Nrf2 gene, spanning from position −1175 to +1240 with the translation initiation site set as position 1. The sequences covered by bisulfite genomic sequencing (−1226 to +844) and contain methylated CpGs are schematically presented with CpG sites indicated by vertical lines. (B) The methylation patterns and extents of CpG sites in the promoter of Nrf2 gene in TRAMP prostate tumor and apparently normal prostate were determined by bisulfite genomic sequencing as described in Material & Methods. Black dots indicate methylated CpGs and open circles indicate non-methylated CpGs. (C) The methylation patterns and extents of CpG sites in the promoter of Nrf2 gene in TRAMP C1 and C3 cells were determined. The CpGs in the sequence between +296 to +594 are not displayed because methylation is insignificant.
Figure 2
Figure 2. Methylation of the first 5 CpGs inhibited the transcriptional activity of Nrf2 promoter.
(A) The construction of luciferase reporters is schematically presented. Nrf2 promoters with (−1367–1) or without (−1065–1) the extra sequence containing the first 5 CpGs were amplified from mouse genomic DNA and inserted into pGL 4.15 vector. The resulted reporters were designated as pGL-1367 and pGL-1065, respectively. (B) pGL-1367 or pGL-1065 reporters, either methylated by CpG methyltransferase or not, were co-transfected with pGL 4.75 vector which contains a Renilla reniformis luciferase gene driven by CMV promoter into TRAMP C1 cells, and the luciferase activities were measured after 24 hrs. The transcriptional activities of each constructs were calculated by normalizing the firefly luciferase activities with corresponding Renilla luciferase activities, and are represented as folds of induction compared with the activity of empty pGL 4.15 vector. The values are mean±SD of four separate samples.
Figure 3
Figure 3. Hypermethylated CpG island was associated with MBD2 binding and histone modifications and 5-aza/TSA treatment reversed the association.
(A) ChIP assay was performed to detect the binding of indicated proteins to specific regions of Nrf2 gene cross-linked and immunoprecipitated from TRAMP C1 and C3 cells. The results from 3 independent experiments were quantified by densitometry as shown in (B). (C) TRAMP C1 cells were treated with vehicle, 5-aza or 5-aza+TSA as described, then the cells were subjected to ChIP assay. The results from 2 independent experiments were quantified by densitometry as shown in (D). ChIP assays were performed as described in Material & Methods using antibodies against Pol II, MBD2, H3K9m3 and AcH3. 3 sets of ChIP primers were used (Table S2), with Nrf2P1 covers the first 5 CpGs (−1190 to −1092) in the CpG island and Nrf2P2 covers a region close to TSS (−62 to +20). Nonspecific IgG was employed as a negative control and binding of Pol II to β-actin promoter was used to verify the efficiency of ChIP assay. The experiments were repeated at least twice with similar results.
Figure 4
Figure 4. The expression of Nrf2 and NQO-1 in TRAMP cell lines was correlated with CpG islands methylation status and could be restored by 5-aza/TSA treatment.
TRAMP C1 and C3 cells were treated with 2 µM 5-aza, 200 nM TSA, or 1 µM 5-aza plus 100 nM TSA for 60 hrs or 48 hrs followed by incubation in the presence of 5 µM tBHQ for further 12 hrs. After treatments, the cells were harvested for total protein or RNA extractions. (A) the mRNA levels of Nrf2 and NQO1 were determined by RT-PCR, with GAPDH serving as internal control, and the results from 3 independent experiments were quantified by densitometry and the results are shown in B and C. (D) the protein levels of Nrf2, NQO1, MBD2, H3K9me3 and H3Ac were determined by Western blotting, with actin as a loading control. Each experiment was repeated at least twice with similar results.
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