Unknown,Transcriptomics,Genomics,Proteomics

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Methylation profiling of human embryonic stems cells and developing human fetal liver reveals distinct dna methylation patterns characterize these samples

ABSTRACT: Publication title: Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver; 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. Experiment Overall Design: lumina gene expression beadchips of human ES cell lines, ES-derived cells, and normal liver (15 samples). High-throughput sequencing of ES cell lines, ES-derived cells, and fetal and normal livers (17 samples). Raw data: SRA008154 http://www.ncbi.nlm.nih.gov/sites/entrez?db=sra&cmd=search&term=SRA008154

ORGANISM(S): Homo sapiens

SUBMITTER: Rami Rauch

PROVIDER: E-GEOD-14966 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Embryogenesis is tightly regulated by multiple levels of epigenetic systems such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent reprogramming occurs by de novo methylation and demethylation. Variance of DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analysed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions in the three germ layers and in the three adult somatic tissues are shared in common. This commonly methylated gene set is enriched in germ cell associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns with global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Taken together, our findings indicate that differentiation from ES cells to the three germ layers is accompanied by an increase in the number of commonly methylated DNA regions and that these tissue-specific alterations are present for only a small number of genes. Our findings indicate that DNA methylation at the proximal promoter regions of commonly methylated genes act as an irreversible mark which fixes somatic lineage by repressing transcription of germ cell specific genes. Using the MeDIP on chip protocol, we immunoprecipitated methylated DNA from R1 ES cell, SK7 cell, as well as SK7 derived-Ectoderm, - Endoderm, -Paraxial mesoderm, brain, liver, skeletal muscle, and sperm, and hybridized to a genome tiling array. The three germ layer lineages from ES cells were confirmed by the expression of specific marker genes in each germ layer (see related expression analysis).

Project description:Hepatocytes that have differentiated from human embryonic stem cells have great potential for the treatment of liver disease as well as for drug testing. Moreover, in vitro hepatogenesis is a powerful model system for studying the molecular mechanisms underlying liver development. DNA methylation is an important epigenetic mechanism that influences differential gene expression during embryonic development. We profiled gene expression and DNA methylation of three cell states of in vitro hepatogenesis—human embryonic stem cells, endoderm progenitors, and mature hepatocytes—using microarray analysis. Among 525 state-specific expressed genes, 67 showed significant negative correlation between gene expression and DNA methylation. State-specific expression and methylation of target genes were validated by quantitative reverse transcription–PCR and pyrosequencing, respectively. To elucidate genome-scale methylation changes beyond the promoter, we also performed high-throughput sequencing of methylated DNA captured by MBD2 protein [see SRA link below]. We found dynamic methylation changes in intergenic regions of the human genome during differentiation. Conclusion: This study provides valuable methylation markers for the lineage commitment of in vitro hepatogenesis and should help elucidate the molecular mechanisms underlying stem cell differentiation and liver development. ES, EP, and MH methylation assays were run in triplicate. Genomic DNA (1 µg) from each sample was bisulfite converted using the EZ DNA methylation kit (Zymo Research, Orange, CA), and 200 ng of the converted DNA was used for amplification. Amplified DNA was hybridized to the Infinium HumanMethylation27 BeadChip (Illumina), which analyzes the methylation status of 27,578 CpG sites selected from more than 14,495 well-annotated genes. The arrays were imaged using the BeadArray Reader, and image processing and extraction of intensity data were performed according to Illumina’s instructions. Each methylation signal was used to compute the β value, which is a quantitative measure of DNA methylation ranging from 0 (for completely unmethylated cytosines) to 1 (for completely methylated cytosines).

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Methylation profiling of human embryonic stems cells and developing human fetal liver reveals distinct dna methylation patterns characterize these samples

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