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Global analysis of DNA methylation in early-stage liver fibrosis.

Komatsu Y, Waku T, Iwasaki N, Ono W, Yamaguchi C, Yanagisawa J - BMC Med Genomics (2012)

Bottom Line: To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and in-vitro-methylation assays.Moreover, Spp1 is also known to enhance tumor development.Using the web-based database, we revealed that Spp1 expression is increased in HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan.

ABSTRACT

Background: Liver fibrosis is caused by chemicals or viral infection. The progression of liver fibrosis results in hepatocellular carcinogenesis in later stages. Recent studies have revealed the importance of DNA hypermethylation in the progression of liver fibrosis to hepatocellular carcinoma (HCC). However, the importance of DNA methylation in the early-stage liver fibrosis remains unclear.

Methods: To address this issue, we used a pathological mouse model of early-stage liver fibrosis that was induced by treatment with carbon tetrachloride (CCl4) for 2 weeks and performed a genome-wide analysis of DNA methylation status. This global analysis of DNA methylation was performed using a combination of methyl-binding protein (MBP)-based high throughput sequencing (MBP-seq) and bioinformatic tools, IPA and Oncomine. To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and in-vitro-methylation assays.

Results: The genome-wide analysis revealed that DNA methylation status was reduced throughout the genome because of CCl4 treatment in the early-stage liver fibrosis. Bioinformatic and biochemical analyses revealed that a gene associated with fibrosis, secreted phosphoprotein 1 (Spp1), which induces inflammation, was hypomethylated and its expression was up-regulated. These results suggest that DNA hypomethylation of the genes responsible for fibrosis may precede the onset of liver fibrosis. Moreover, Spp1 is also known to enhance tumor development. Using the web-based database, we revealed that Spp1 expression is increased in HCC.

Conclusions: Our study suggests that hypomethylation is crucial for the onset of and in the progression of liver fibrosis to HCC. The elucidation of this change in methylation status from the onset of fibrosis and subsequent progression to HCC may lead to a new clinical diagnosis.

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Validation of the methylation site located upstream of the Spp1 TSS. (A) Epigenetic features of the methylation site located upstream of the Spp1 TSS assessed using the UCSC Genome browser. The Spp1 gene body is shown in dark blue. The methylation site upstream of the Spp1 TSS is shown as a red rectangle on the chromosome diagram and on the custom track. ChIP-seq signals for H3K4 me1 and H3K4 me4 in the liver (orange), for RNAP II in the liver and p300 in MEL leukemia (light blue), and for the DNase I hotspot in the liver (green) are represented as density plots. (B) MBP-IP assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents fold changes plus the standard deviation (**p-value < 0.01, n = 3-4). (C) Bisulfite modification assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents percentage of methylated sites in the site located upstream of the Spp1 TSS (n = 10).
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Figure 4: Validation of the methylation site located upstream of the Spp1 TSS. (A) Epigenetic features of the methylation site located upstream of the Spp1 TSS assessed using the UCSC Genome browser. The Spp1 gene body is shown in dark blue. The methylation site upstream of the Spp1 TSS is shown as a red rectangle on the chromosome diagram and on the custom track. ChIP-seq signals for H3K4 me1 and H3K4 me4 in the liver (orange), for RNAP II in the liver and p300 in MEL leukemia (light blue), and for the DNase I hotspot in the liver (green) are represented as density plots. (B) MBP-IP assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents fold changes plus the standard deviation (**p-value < 0.01, n = 3-4). (C) Bisulfite modification assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents percentage of methylated sites in the site located upstream of the Spp1 TSS (n = 10).

Mentions: The hypomethylation peak was located approximately 18 kbp upstream of the transcription start site (TSS) of Spp1 (Figure 4A, red). We placed this MBP-seq peak into the UCSC genome browser and found that the hypomethylation site upstream of Spp1 has several chromatin features identified from studies by other groups, including: 1) mono- and trimethylation of histone 3 lysine 4 in the liver (H3K4 me1 and H3K4 me3) (Figure 4A, orange) [20]; 2) the binding of RNA polymerase II and related acetyltransferace p300 in the liver and in the MEL leukemia cell line (Figure 4A, light blue) [21]; and 3) it is a DNase I hotspot in the liver (Figure 4A, green) [22]. Previous studies have demonstrated that these epigenetic features are observed in the enhancer region [20,23-25], and that hypomethylation of an enhancer predominantly induces mRNA expression [26-28]. Thus, these epigenetic annotations lead to the hypothesis that this hypomethylated site may function as an enhancer that regulates Spp1 expression.


Global analysis of DNA methylation in early-stage liver fibrosis.

Komatsu Y, Waku T, Iwasaki N, Ono W, Yamaguchi C, Yanagisawa J - BMC Med Genomics (2012)

Validation of the methylation site located upstream of the Spp1 TSS. (A) Epigenetic features of the methylation site located upstream of the Spp1 TSS assessed using the UCSC Genome browser. The Spp1 gene body is shown in dark blue. The methylation site upstream of the Spp1 TSS is shown as a red rectangle on the chromosome diagram and on the custom track. ChIP-seq signals for H3K4 me1 and H3K4 me4 in the liver (orange), for RNAP II in the liver and p300 in MEL leukemia (light blue), and for the DNase I hotspot in the liver (green) are represented as density plots. (B) MBP-IP assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents fold changes plus the standard deviation (**p-value < 0.01, n = 3-4). (C) Bisulfite modification assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents percentage of methylated sites in the site located upstream of the Spp1 TSS (n = 10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3295686&req=5

Figure 4: Validation of the methylation site located upstream of the Spp1 TSS. (A) Epigenetic features of the methylation site located upstream of the Spp1 TSS assessed using the UCSC Genome browser. The Spp1 gene body is shown in dark blue. The methylation site upstream of the Spp1 TSS is shown as a red rectangle on the chromosome diagram and on the custom track. ChIP-seq signals for H3K4 me1 and H3K4 me4 in the liver (orange), for RNAP II in the liver and p300 in MEL leukemia (light blue), and for the DNase I hotspot in the liver (green) are represented as density plots. (B) MBP-IP assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents fold changes plus the standard deviation (**p-value < 0.01, n = 3-4). (C) Bisulfite modification assay of the methylation status of the site located upstream of the Spp1 TSS. The CCl4 sample was normalized to the control sample. The vertical axis represents percentage of methylated sites in the site located upstream of the Spp1 TSS (n = 10).
Mentions: The hypomethylation peak was located approximately 18 kbp upstream of the transcription start site (TSS) of Spp1 (Figure 4A, red). We placed this MBP-seq peak into the UCSC genome browser and found that the hypomethylation site upstream of Spp1 has several chromatin features identified from studies by other groups, including: 1) mono- and trimethylation of histone 3 lysine 4 in the liver (H3K4 me1 and H3K4 me3) (Figure 4A, orange) [20]; 2) the binding of RNA polymerase II and related acetyltransferace p300 in the liver and in the MEL leukemia cell line (Figure 4A, light blue) [21]; and 3) it is a DNase I hotspot in the liver (Figure 4A, green) [22]. Previous studies have demonstrated that these epigenetic features are observed in the enhancer region [20,23-25], and that hypomethylation of an enhancer predominantly induces mRNA expression [26-28]. Thus, these epigenetic annotations lead to the hypothesis that this hypomethylated site may function as an enhancer that regulates Spp1 expression.

Bottom Line: To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and in-vitro-methylation assays.Moreover, Spp1 is also known to enhance tumor development.Using the web-based database, we revealed that Spp1 expression is increased in HCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan.

ABSTRACT

Background: Liver fibrosis is caused by chemicals or viral infection. The progression of liver fibrosis results in hepatocellular carcinogenesis in later stages. Recent studies have revealed the importance of DNA hypermethylation in the progression of liver fibrosis to hepatocellular carcinoma (HCC). However, the importance of DNA methylation in the early-stage liver fibrosis remains unclear.

Methods: To address this issue, we used a pathological mouse model of early-stage liver fibrosis that was induced by treatment with carbon tetrachloride (CCl4) for 2 weeks and performed a genome-wide analysis of DNA methylation status. This global analysis of DNA methylation was performed using a combination of methyl-binding protein (MBP)-based high throughput sequencing (MBP-seq) and bioinformatic tools, IPA and Oncomine. To confirm functional aspect of MBP-seq data, we complementary used biochemical methods, such as bisulfite modification and in-vitro-methylation assays.

Results: The genome-wide analysis revealed that DNA methylation status was reduced throughout the genome because of CCl4 treatment in the early-stage liver fibrosis. Bioinformatic and biochemical analyses revealed that a gene associated with fibrosis, secreted phosphoprotein 1 (Spp1), which induces inflammation, was hypomethylated and its expression was up-regulated. These results suggest that DNA hypomethylation of the genes responsible for fibrosis may precede the onset of liver fibrosis. Moreover, Spp1 is also known to enhance tumor development. Using the web-based database, we revealed that Spp1 expression is increased in HCC.

Conclusions: Our study suggests that hypomethylation is crucial for the onset of and in the progression of liver fibrosis to HCC. The elucidation of this change in methylation status from the onset of fibrosis and subsequent progression to HCC may lead to a new clinical diagnosis.

Show MeSH
Related in: MedlinePlus