DNA methylation profiling of embryonic stem cell differentiation into the three germ layers

Abstract

Embryogenesis is tightly regulated by multiple levels of epigenetic regulation 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 developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed 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 are methylated across all three germ layers and in the three adult somatic tissues. 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 by 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. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes.

Figure 1. Defining the promoter methylome.

a) Microarray detection of DNA hypermethylation on imprinting center regions (ICRs). A green line indicates a CpG island region. A blue line indicates an ICR. b) (left panel) Scatter plots show the DNA methylation levels for all probes relative to their CpG content (CpG/bp). Each spot represents one probe. (right panel) Quantification of DNA methylation for a subset of probes (left panel). The red line indicates the region amplified for bisulfite sequencing. CpGs are represented as open dots (if unmethylated) or filled dots (if methylated). The percentage of CpG methylation is indicated for each amplicon. c) The MATscore distribution of array regions corresponding to INPUT (red) and MeDIP (blue). d) The FDR (%) distribution corresponding to each MATscore.

Figure 2. DNA methylation at transcription start sites (TSSs).

a) DNA methylation frequency relative to the distance to the TSS. Methylation frequency was calculated using the following formula: Number of methylated probes/Number of total probes at each position from the TSS. The line indicates the moving average. b) Expression levels of genes associated with CMRs in each region around the TSS. The core region shows the region between 1 kb upstream and 0.5 kb downstream of the TSS. Up, indicates 1 kbP<0.01, a t-test.).

Figure 3. Testis specific genes are commonly methylated in somatic lineages.

a) Overview of DNA methylation profiling b) Methylation of germline-specific genes in somatic lineages. A green line indicates a CpG island region. c) Gene Set Enrichment Analysis (GSEA) determines whether a defined set of genes shows concordant changes between two biological states. The normalized enrichment score (NES) reflects the degree to which a gene set is upregulated (positive NES). Corresponding p values are indicated.

Figure 4. Methylation changes in cells derived from mouse ES cells and mouse adult tissues.

a) Overview of variable DNA methylation profiles among the three germ layers. The arrowhead indicates ectodermal relaxation of DNA methylation of Igf2r. b) DNA methylation profile and schematic representation of the Igf2r imprinting region. A green line indicates a CpG island region. A blue line indicates an imprinting center region (ICR). c) Validation of DNA methylation in the Igf2r imprinting region. (i) indicates the normal imprinting region, and (ii) indicates the ectodermal relaxation of DNA methylation. The DNA methylation level was quantitatively estimated using MALDI/TOFMS. d) Expression levels of variable methylation-associated genes for each sample. Pair-wise comparisons of expression levels of methylated or hypomethylated genes were performed using a t-test.

Figure 5. The Pcdh cluster is methylated during differentiation into the three germ layers.

a) Overview of the DNA methylation profile of the Pcdh-γ cluster. A green line indicates a CpG island region. The arrowhead indicates the region analyzed for DNA methylation. b) Bisulfite sequencing analysis of the DNA methylation status of the first exon of Pcdh-γα2. c) Gene expression pattern of Pcdh-γα2.

Figure 6. The Rhox cluster is classified into two regulated regions by DNA methylation.

a) Overview of the DNA methylation profile of the Rhox cluster. b) The DNA methylation status of anterior and posterior Rhox genes. The left panel represents the MeDIP signal in each Rhox promoter. The right panel shows the quantitative estimation of DNA methylation by MALDI/TOFMAS. The arrowhead indicates the region of DNA analyzed for methylation. c) Expression profile of anterior and posterior Rhox genes.

Figure 7. The exclusive relationship between DNA methylation and histone methylation.

a) Representation of DNA methylation, histone methylation and gene expression. b) Percentage of genes DNA methylated with each histone modification. c) The percentage of DNA methylation in promoters is conditional on the histone methylation state in ES (Sk7_ESC) and NPC/Ect cells and in brain (n, number of promoters).