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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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Quantitative Chromatin Immunoprecipitation (ChIP) assay of the MT3 promoter.The three ChIP assays of the MT3 promoter are shown in Figure 1A and Table 1. All ChIP experiments were performed in triplicates for each assay. Following quantitative real-time PCR (qPCR), the percent of relative of DNA binding in B, C and D was determined by normalizing the indicated histone binding level to its corresponding input and H3 levels. A relative percent of occupancy of three histones is shown. A) DNA methylation levels in the CpG nucleotides from −161 to +344 in OE33 and FLO-1 cell lines demonstrate a lower methylation level in FLO-1 cells. B) ChIP-qPCR in FLO-1 cells, demonstrate relatively low levels of repressive histone H3K9me2 as compared to histone H3K4me2 and H3K9ace. C) ChIP-qPCR results in the OE33 cell line show high levels of H3K9me2 as compared to the other two histones. D) ChIP-qPCR results in OE33 cell line, following administration of 5 µM 5-Aza for 72 h, reversed the relative abundance of the histone modifications towards active expression.
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pone-0022009-g005: Quantitative Chromatin Immunoprecipitation (ChIP) assay of the MT3 promoter.The three ChIP assays of the MT3 promoter are shown in Figure 1A and Table 1. All ChIP experiments were performed in triplicates for each assay. Following quantitative real-time PCR (qPCR), the percent of relative of DNA binding in B, C and D was determined by normalizing the indicated histone binding level to its corresponding input and H3 levels. A relative percent of occupancy of three histones is shown. A) DNA methylation levels in the CpG nucleotides from −161 to +344 in OE33 and FLO-1 cell lines demonstrate a lower methylation level in FLO-1 cells. B) ChIP-qPCR in FLO-1 cells, demonstrate relatively low levels of repressive histone H3K9me2 as compared to histone H3K4me2 and H3K9ace. C) ChIP-qPCR results in the OE33 cell line show high levels of H3K9me2 as compared to the other two histones. D) ChIP-qPCR results in OE33 cell line, following administration of 5 µM 5-Aza for 72 h, reversed the relative abundance of the histone modifications towards active expression.

Mentions: FLO-1 and OE33 cell lines were selected for the ChIP assays, based on their variable methylation and expression levels of MT3. OE33 cells have a high level of DNA methylation (74% for Region 2) and almost silenced MT3 expression, whereas FLO-1 cells express MT3 and have a low level of DNA methylation (9% for Region 2) (Figure 5A). To examine if the DNA hypermethylation of the MT3 gene promoter region is associated with additional repressor histone modifications, the chromatin immunoprecipitation assay (ChIP) was carried out using specific antibodies against H3, H3K4 dimethylation (H3K4me2), H3K9 dimethylation (H3K9me2), and H3K9 acetylation (H3K9ace). Three independent assays were developed along different regions of the MT3 promoter as shown in Figure 1A and Table 1. FLO-1 cells showed a very low level of repressive histone H3K9me2 as compared to histone H3K4me2 and H3K9ace (Figure 5B). In contrast, the OE33 cells displayed a significant level of H3K9me2 as compared to the other two histones (Figure 5C). Administration of 5-Aza (5 uM) to OE33 cells for 72 h reversed the relative abundance of the histone modifications towards active expression. There was a decrease in the level of H3K9me2 to MT3 promoter regions and an increase in H3K4ac and H3K4me2 (Figure 5D).


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)

Quantitative Chromatin Immunoprecipitation (ChIP) assay of the MT3 promoter.The three ChIP assays of the MT3 promoter are shown in Figure 1A and Table 1. All ChIP experiments were performed in triplicates for each assay. Following quantitative real-time PCR (qPCR), the percent of relative of DNA binding in B, C and D was determined by normalizing the indicated histone binding level to its corresponding input and H3 levels. A relative percent of occupancy of three histones is shown. A) DNA methylation levels in the CpG nucleotides from −161 to +344 in OE33 and FLO-1 cell lines demonstrate a lower methylation level in FLO-1 cells. B) ChIP-qPCR in FLO-1 cells, demonstrate relatively low levels of repressive histone H3K9me2 as compared to histone H3K4me2 and H3K9ace. C) ChIP-qPCR results in the OE33 cell line show high levels of H3K9me2 as compared to the other two histones. D) ChIP-qPCR results in OE33 cell line, following administration of 5 µM 5-Aza for 72 h, reversed the relative abundance of the histone modifications towards active expression.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139601&req=5

pone-0022009-g005: Quantitative Chromatin Immunoprecipitation (ChIP) assay of the MT3 promoter.The three ChIP assays of the MT3 promoter are shown in Figure 1A and Table 1. All ChIP experiments were performed in triplicates for each assay. Following quantitative real-time PCR (qPCR), the percent of relative of DNA binding in B, C and D was determined by normalizing the indicated histone binding level to its corresponding input and H3 levels. A relative percent of occupancy of three histones is shown. A) DNA methylation levels in the CpG nucleotides from −161 to +344 in OE33 and FLO-1 cell lines demonstrate a lower methylation level in FLO-1 cells. B) ChIP-qPCR in FLO-1 cells, demonstrate relatively low levels of repressive histone H3K9me2 as compared to histone H3K4me2 and H3K9ace. C) ChIP-qPCR results in the OE33 cell line show high levels of H3K9me2 as compared to the other two histones. D) ChIP-qPCR results in OE33 cell line, following administration of 5 µM 5-Aza for 72 h, reversed the relative abundance of the histone modifications towards active expression.
Mentions: FLO-1 and OE33 cell lines were selected for the ChIP assays, based on their variable methylation and expression levels of MT3. OE33 cells have a high level of DNA methylation (74% for Region 2) and almost silenced MT3 expression, whereas FLO-1 cells express MT3 and have a low level of DNA methylation (9% for Region 2) (Figure 5A). To examine if the DNA hypermethylation of the MT3 gene promoter region is associated with additional repressor histone modifications, the chromatin immunoprecipitation assay (ChIP) was carried out using specific antibodies against H3, H3K4 dimethylation (H3K4me2), H3K9 dimethylation (H3K9me2), and H3K9 acetylation (H3K9ace). Three independent assays were developed along different regions of the MT3 promoter as shown in Figure 1A and Table 1. FLO-1 cells showed a very low level of repressive histone H3K9me2 as compared to histone H3K4me2 and H3K9ace (Figure 5B). In contrast, the OE33 cells displayed a significant level of H3K9me2 as compared to the other two histones (Figure 5C). Administration of 5-Aza (5 uM) to OE33 cells for 72 h reversed the relative abundance of the histone modifications towards active expression. There was a decrease in the level of H3K9me2 to MT3 promoter regions and an increase in H3K4ac and H3K4me2 (Figure 5D).

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