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Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas.

Peng D, Hu TL, Jiang A, Washington MK, Moskaluk CA, Schneider-Stock R, El-Rifai W - PLoS ONE (2011)

Bottom Line: Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively).The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells.The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT

Background: Metallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs).

Methods and results: Using quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from -372 to -306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (-4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from -139 to -49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, -log10(FDR)>3.0]. The DNA methylation levels from -127 to -8 CpG sites showed the strongest correlation with MT3 gene expression (r = -0.4, P<0.0001). Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks.

Conclusion: In summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

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Related in: MedlinePlus

DNA methylation levels of the MT3 promoter in esophageal adenocarcinomas and normal tissue samples.A) Schematic illustration of MT3 genomic structure and the Pyrosequencing and ChIP assays. The MT3 gene comprises of 3 exons (black boxes). There is a long CpG island extending from upstream of the transcription start site (TSS) to the first intron (grey box). Each grey vertical bar represents one individual CpG site from −372 to +344. Four Pyrosequencing assays (assay 1–4) and three ChIP assays (ChIP 1–3) are shown. B) Heat map of DNA methylation profile from −372 to +344 in normal esophageal squamous mucosa (NS), normal gastric glandular mucosa (NG), Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) samples. A key for the blue scale is given below the map. Grey areas indicate missing data. C) Significance analysis of DNA methylation levels in the MT3 promoter region from −161 to +344 of TSS between EAC and normal samples. Each dot or cycle represents one comparison between tumor and normal samples for one CpG site, as indicated. The graph is plotted for the mean value in all tumor samples, as compared to the mean value for all normal samples (NS or NG) in each CpG site. FDR (false discovery rate) was used to demonstrate the significance and is plotted as −log10(FDR) in y axis. Thus, if FDR<0.01 then −log10(FDR)>2.0. We used FDR≤0.01 as the cutoff for statistical significance.
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pone-0022009-g001: DNA methylation levels of the MT3 promoter in esophageal adenocarcinomas and normal tissue samples.A) Schematic illustration of MT3 genomic structure and the Pyrosequencing and ChIP assays. The MT3 gene comprises of 3 exons (black boxes). There is a long CpG island extending from upstream of the transcription start site (TSS) to the first intron (grey box). Each grey vertical bar represents one individual CpG site from −372 to +344. Four Pyrosequencing assays (assay 1–4) and three ChIP assays (ChIP 1–3) are shown. B) Heat map of DNA methylation profile from −372 to +344 in normal esophageal squamous mucosa (NS), normal gastric glandular mucosa (NG), Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) samples. A key for the blue scale is given below the map. Grey areas indicate missing data. C) Significance analysis of DNA methylation levels in the MT3 promoter region from −161 to +344 of TSS between EAC and normal samples. Each dot or cycle represents one comparison between tumor and normal samples for one CpG site, as indicated. The graph is plotted for the mean value in all tumor samples, as compared to the mean value for all normal samples (NS or NG) in each CpG site. FDR (false discovery rate) was used to demonstrate the significance and is plotted as −log10(FDR) in y axis. Thus, if FDR<0.01 then −log10(FDR)>2.0. We used FDR≤0.01 as the cutoff for statistical significance.

Mentions: Our analysis indicated that the human MT3 gene contains a large CpG island located from −372 bp upstream of the transcript start site (TSS) to approximately +344 bp downstream of the TSS (Figure 1A). To determine the DNA methylation change of the CpG island, we designed several Pyrosequencing assays that enabled us to perform quantitative analysis of the DNA methylation level of each of the 59 CpG nucleotides using a state-of-the-art Pyrosequencing technique (Figure 1A). A heat map of the DNA methylation levels of each of the CpG sites from −372 to +344 of the TSS in NS (normal esophageal squamous epithelia), NG (normal gastric epithelia), BE, and EAC is shown in Figure 1B. A distant upstream region of CpG nucleotides from −372 to −306 displayed non-exclusive high level DNA methylation in all the normal and tumor samples (The 6 CpG sites at the left side in Figure 1B). This finding suggests that this region may not play a critical role in transcription regulation of MT3 during EAC carcinogenesis, and was excluded from further analysis. On the other hand, the normal samples (both NS and NG) displayed universal low levels of DNA methylation in the remaining CpG sites (Figure 1B), suggesting that the “field defect” phenomenon may not play a significant role in the regulation of MT3 gene expression. There was a significant difference in the DNA methylation level between EACs and both normal esophagus (NS) and normal stomach (NG) samples in almost all CpG nucleotides in the region from −161 to +344 (FDR<0.01, −log10(FDR)>2, Figure 1C). However, CpG nucleotides in two regions (−139 to −49 and +296 to +344) were more significant than all other sites (FDR<0.001, −log10(FDR)>3.0, Figure 1C). Interestingly, DNA methylation changes in this region displayed a biphasic methylation pattern in primary EACs: declining to <10% towards the TSS as seen in CpG nucleotides at −4 and +3 of TSS (Figure 2A). In our analysis, we divided the region from −161 to +344 into 3 subregions. The CpG nucleotides from −161 to −130 of the TSS (Region 1, R1) displayed increased DNA methylation in normal samples (10–25%). The methylation levels of these sites in normal stomach samples (NG) were significantly higher than in normal esophagus (NS, P<0.01). BE and EAC samples had higher DNA methylation levels than in normal samples (P<0.01 among N, BE, EAC, and between them, Figure 2B). The CpG nucleotides from −127 to −8 (Region 2, R2) demonstrated low methylation levels (below 10%) in both normal esophagus and stomach with a significant increase in the methylation levels in BE and EACs (P<0.001 among N, BE, EAC, Figure 2B). Similarly, the CpG nucleotides from +28 and +344 (Region 3, R3) showed low methylation levels in normal esophagus and stomach samples. However, this region showed similar higher methylation levels in BE and EACs, in particular, in sites from +28 to +277 (Figure 2A).


Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas.

Peng D, Hu TL, Jiang A, Washington MK, Moskaluk CA, Schneider-Stock R, El-Rifai W - PLoS ONE (2011)

DNA methylation levels of the MT3 promoter in esophageal adenocarcinomas and normal tissue samples.A) Schematic illustration of MT3 genomic structure and the Pyrosequencing and ChIP assays. The MT3 gene comprises of 3 exons (black boxes). There is a long CpG island extending from upstream of the transcription start site (TSS) to the first intron (grey box). Each grey vertical bar represents one individual CpG site from −372 to +344. Four Pyrosequencing assays (assay 1–4) and three ChIP assays (ChIP 1–3) are shown. B) Heat map of DNA methylation profile from −372 to +344 in normal esophageal squamous mucosa (NS), normal gastric glandular mucosa (NG), Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) samples. A key for the blue scale is given below the map. Grey areas indicate missing data. C) Significance analysis of DNA methylation levels in the MT3 promoter region from −161 to +344 of TSS between EAC and normal samples. Each dot or cycle represents one comparison between tumor and normal samples for one CpG site, as indicated. The graph is plotted for the mean value in all tumor samples, as compared to the mean value for all normal samples (NS or NG) in each CpG site. FDR (false discovery rate) was used to demonstrate the significance and is plotted as −log10(FDR) in y axis. Thus, if FDR<0.01 then −log10(FDR)>2.0. We used FDR≤0.01 as the cutoff for statistical significance.
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Related In: Results  -  Collection

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pone-0022009-g001: DNA methylation levels of the MT3 promoter in esophageal adenocarcinomas and normal tissue samples.A) Schematic illustration of MT3 genomic structure and the Pyrosequencing and ChIP assays. The MT3 gene comprises of 3 exons (black boxes). There is a long CpG island extending from upstream of the transcription start site (TSS) to the first intron (grey box). Each grey vertical bar represents one individual CpG site from −372 to +344. Four Pyrosequencing assays (assay 1–4) and three ChIP assays (ChIP 1–3) are shown. B) Heat map of DNA methylation profile from −372 to +344 in normal esophageal squamous mucosa (NS), normal gastric glandular mucosa (NG), Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) samples. A key for the blue scale is given below the map. Grey areas indicate missing data. C) Significance analysis of DNA methylation levels in the MT3 promoter region from −161 to +344 of TSS between EAC and normal samples. Each dot or cycle represents one comparison between tumor and normal samples for one CpG site, as indicated. The graph is plotted for the mean value in all tumor samples, as compared to the mean value for all normal samples (NS or NG) in each CpG site. FDR (false discovery rate) was used to demonstrate the significance and is plotted as −log10(FDR) in y axis. Thus, if FDR<0.01 then −log10(FDR)>2.0. We used FDR≤0.01 as the cutoff for statistical significance.
Mentions: Our analysis indicated that the human MT3 gene contains a large CpG island located from −372 bp upstream of the transcript start site (TSS) to approximately +344 bp downstream of the TSS (Figure 1A). To determine the DNA methylation change of the CpG island, we designed several Pyrosequencing assays that enabled us to perform quantitative analysis of the DNA methylation level of each of the 59 CpG nucleotides using a state-of-the-art Pyrosequencing technique (Figure 1A). A heat map of the DNA methylation levels of each of the CpG sites from −372 to +344 of the TSS in NS (normal esophageal squamous epithelia), NG (normal gastric epithelia), BE, and EAC is shown in Figure 1B. A distant upstream region of CpG nucleotides from −372 to −306 displayed non-exclusive high level DNA methylation in all the normal and tumor samples (The 6 CpG sites at the left side in Figure 1B). This finding suggests that this region may not play a critical role in transcription regulation of MT3 during EAC carcinogenesis, and was excluded from further analysis. On the other hand, the normal samples (both NS and NG) displayed universal low levels of DNA methylation in the remaining CpG sites (Figure 1B), suggesting that the “field defect” phenomenon may not play a significant role in the regulation of MT3 gene expression. There was a significant difference in the DNA methylation level between EACs and both normal esophagus (NS) and normal stomach (NG) samples in almost all CpG nucleotides in the region from −161 to +344 (FDR<0.01, −log10(FDR)>2, Figure 1C). However, CpG nucleotides in two regions (−139 to −49 and +296 to +344) were more significant than all other sites (FDR<0.001, −log10(FDR)>3.0, Figure 1C). Interestingly, DNA methylation changes in this region displayed a biphasic methylation pattern in primary EACs: declining to <10% towards the TSS as seen in CpG nucleotides at −4 and +3 of TSS (Figure 2A). In our analysis, we divided the region from −161 to +344 into 3 subregions. The CpG nucleotides from −161 to −130 of the TSS (Region 1, R1) displayed increased DNA methylation in normal samples (10–25%). The methylation levels of these sites in normal stomach samples (NG) were significantly higher than in normal esophagus (NS, P<0.01). BE and EAC samples had higher DNA methylation levels than in normal samples (P<0.01 among N, BE, EAC, and between them, Figure 2B). The CpG nucleotides from −127 to −8 (Region 2, R2) demonstrated low methylation levels (below 10%) in both normal esophagus and stomach with a significant increase in the methylation levels in BE and EACs (P<0.001 among N, BE, EAC, Figure 2B). Similarly, the CpG nucleotides from +28 and +344 (Region 3, R3) showed low methylation levels in normal esophagus and stomach samples. However, this region showed similar higher methylation levels in BE and EACs, in particular, in sites from +28 to +277 (Figure 2A).

Bottom Line: Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively).The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells.The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT

Background: Metallothionein 3 (MT3) maintains intracellular metal homeostasis and protects against reactive oxygen species (ROS)-induced DNA damage. In this study, we investigated the epigenetic alterations and gene expression of the MT3 gene in esophageal adenocarcinomas (EACs).

Methods and results: Using quantitative bisulfite pyrosequencing, we detected unique DNA methylation profiles in the MT3 promoter region. The CpG nucleotides from -372 to -306 from the transcription start site (TSS) were highly methylated in tumor (n = 64) and normal samples (n = 51), whereas CpG nucleotides closest to the TSS (-4 and +3) remained unmethylated in all normal and most tumor samples. Conversely, CpG nucleotides in two regions (from -139 to -49 and +296 to +344) were significantly hypermethylated in EACs as compared to normal samples [FDR<0.001, -log10(FDR)>3.0]. The DNA methylation levels from -127 to -8 CpG sites showed the strongest correlation with MT3 gene expression (r = -0.4, P<0.0001). Moreover, the DNA hypermethylation from -127 to -8 CpG sites significantly correlated with advanced tumor stages and lymph node metastasis (P = 0.005 and P = 0.0313, respectively). The ChIP analysis demonstrated a more repressive histone modification (H3K9me2) and less active histone modifications (H3K4me2, H3K9ace) in OE33 cells than in FLO-1 cells; concordant with the presence of higher DNA methylation levels and silencing of MT3 expression in OE33 as compared to FLO-1 cells. Treatment of OE33 cells with 5-Aza-deoxycitidine restored MT3 expression with demethylation of its promoter region and reversal of the histone modifications towards active histone marks.

Conclusion: In summary, EACs are characterized by frequent epigenetic silencing of MT3. The choice of specific regions in the CpG island is a critical step in determining the functional role and prognostic value of DNA methylation in cancer cells.

Show MeSH
Related in: MedlinePlus