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Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver.

ABSTRACT: To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%). Second, in vitro hESC differentiation is characterized by both de novo methylation and demethylation, whereas in vivo fetal liver development is characterized predominantly by demethylation. Third, hESC differentiation is uniquely characterized by methylation changes specifically at H3K27me3-occupied regions, bivalent domains, and low density CpG promoters (LCPs), suggesting that these regions are more likely to be involved in transcriptional regulation during hESC differentiation. Although both H3K27me3-occupied domains and LCPs are also regions of high variability in DNA methylation state during human liver development, these regions become highly unmethylated, which is a distinct trend from that observed in hESCs. Taken together, our results indicate that hESC differentiation has a unique DNA methylation signature that may not be indicative of in vivo differentiation.

SUBMITTER: Brunner AL

PROVIDER: S-EPMC2694474 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver.

Brunner Alayne L AL Johnson David S DS Kim Si Wan SW Valouev Anton A Reddy Timothy E TE Neff Norma F NF Anton Elizabeth E Medina Catherine C Nguyen Loan L Chiao Eric E Oyolu Chuba B CB Schroth Gary P GP Absher Devin M DM Baker Julie C JC Myers Richard M RM

Genome research 20090309 6

To investigate the role of DNA methylation during human development, we developed Methyl-seq, a method that assays DNA methylation at more than 90,000 regions throughout the genome. Performing Methyl-seq on human embryonic stem cells (hESCs), their derivatives, and human tissues allowed us to identify several trends during hESC and in vivo liver differentiation. First, differentiation results in DNA methylation changes at a minimal number of assayed regions, both in vitro and in vivo (2%-11%). S ...[more]

PMID: 19273619

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Project description:BackgroundDNA methylation is an important epigenetic control mechanism that has been shown to be associated with gene silencing through the course of development, maturation and aging. However, only limited data are available regarding the relationship between methylation and gene expression in human development.ResultsWe analyzed the methylome and transcriptome of three human fetal liver samples (gestational age 20-22 weeks) and three adult human liver samples. Genes whose expression differed between fetal and adult numbered 7,673. Adult overexpression was associated with metabolic pathways and, in particular, cytochrome P450 enzymes while fetal overexpression reflected enrichment for DNA replication and repair. Analysis for DNA methylation using the Illumina Infinium 450 K HumanMethylation BeadChip showed that 42% of the quality filtered 426,154 methylation sites differed significantly between adult and fetal tissue (q ≤ 0.05). Differences were small; 69% of the significant sites differed in their mean methylation beta value by ≤0.2. There was a trend among all sites toward higher methylation in the adult samples with the most frequent difference in beta being 0.1. Characterization of the relationship between methylation and expression revealed a clear difference between fetus and adult. Methylation of genes overexpressed in fetal liver showed the same pattern as seen for genes that were similarly expressed in fetal and adult liver. In contrast, adult overexpressed genes showed fetal hypermethylation that differed from the similarly expressed genes. An examination of gene region-specific methylation showed that sites proximal to the transcription start site or within the first exon with a significant fetal-adult difference in beta (>0.2) showed an inverse relationship with gene expression.ConclusionsNearly half of the CpGs in human liver show a significant difference in methylation comparing fetal and adult samples. Sites proximal to the transcription start site or within the first exon that show a transition from hypermethylation in the fetus to hypomethylation or intermediate methylation in the adult are associated with inverse changes in gene expression. In contrast, increases in methylation going from fetal to adult are not associated with fetal-to-adult decreased expression. These findings indicate fundamentally different roles for and/or regulation of DNA methylation in human fetal and adult liver.

Project description:DNA methylation is an important epigenetic control mechanism that has been shown to be associated with gene silencing through the course of development, maturation and aging. However, only limited data are available regarding the relationship between methylation and gene expression in human development. We analyzed the methylomes and transcriptomes of three human fetal liver samples (gestational age 20-22 weeks) and three adult human liver samples. Genes whose expression differed between fetal and adult numbered 7,673. Adult overexpression was associated with metabolic pathways and, in particular, cytochrome P450 enzymes while fetal overexpression reflected enrichment for DNA replication and repair. Analysis for DNA methylation using the Illumina Infinium 450K HumanMethylation BeadChip showed that 42% of the quality filtered 426,154 methylation sites differed significantly between adult and fetal tissue (q≤0.05). Differences were small; 69% of the significant sites differed in their mean methylation beta value by ≤0.2. There was a trend among all sites toward higher methylation in the adult samples with the most frequent difference in beta being 0.1. Characterization of the relationship between methylation and expression revealed a clear difference between fetus and adult. Methylation of genes overexpressed in fetal liver showed the same pattern as seen for genes that were similarly expressed in fetal and adult liver. In contrast, adult overexpressed genes showed fetal hypermethylation that differed from the similarly expressed genes. An examination of gene region-specific methylation showed that sites proximal to the transcription start site or within the first exon with a significant fetal-adult difference in beta (>0.2) showed an inverse relationship with gene expression. Nearly half of the CpGs in human liver show a significant difference in methylation comparing fetal and adult samples. Sites proximal to the transcription start site or within the first exon that show a transition from hypermethylation in the fetus to hypomethylation or intermediate methylation in the adult are associated with inverse changes in gene expression. In contrast, increases in methylation going from fetal to adult are not associated with fetal-to-adult decreased expression. These findings indicate fundamentally different roles for and/or regulation of DNA methylation in human fetal and adult liver.

Project description:Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3-FOXO1 or PAX7-FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3-FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3-FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2'-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

Dataset Information

Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver.

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Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver.

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