DNA methylation programming and reprogramming in primate embryonic stem cells - PubMed (original) (raw)

DNA methylation programming and reprogramming in primate embryonic stem cells

Netta Mendelson Cohen et al. Genome Res. 2009 Dec.

Abstract

DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem cells (ESCs), fibroblasts, and ESCs generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper- or hypomethylated in fibroblasts compared to native ESCs and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ESCs, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.

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Figures

Figure 1.

Figure 1.

Profiling monkey ES methylation. (A) Experimental design. We studied DNA methylation in native ESCs (ORMES-22), fibroblasts, and ESCs generated by somatic cell nuclear transfer (CRES-2). We also assayed a distinctly different native ES line (the homozygous parthenote ORMES-9) to control for ES line-specific effects. DNA methylation profiles in these four cell types were assayed using MeDIP and tiling arrays. Values of zero correspond to average genomic methylation. (B) Near perfect DNA methylation reprogramming in CRES-2. Shown are the differential methylation values for 380,000 array probes covering orthologous human K4–K27 bivalent domains and selected DNA methylation hotspots. The differences between fibroblasts and the two stem cell lines are highly correlated, showing that at the global level, reprogramming of the fibroblast epigenome during nuclear transfer is near perfect. (C) Conserved and differential methylation in HOX clusters. Shown are the methylation profiles at the (upper) HOXA and (lower) HOXD clusters, which were tiled completely on our array and reflect an excellent overall correlation between the native and reprogrammed ESCs. Regions undergoing fibroblast (red) hypermethylation (hyper-DMRs) or (green) hypomethylation (hypo-DMRs) are highlighted. In contrast to the good overall correspondence between native and reprogrammed ESC methylation, a (blue) small region in the HOXA cluster shows a CRES-2 methylation pattern that is similar to the fibroblast profile, suggesting incomplete reprogramming or independent hypomethylation in OMRES-22 and ORMES-9.

Figure 2.

Figure 2.

Differentially methylated regions (DMRs). (A) Global patterns of methylation reprogramming. DMRs were statistically extracted from the data by comparing methylation in all pairs of cell types, thereby not pre-assuming any type of organization. Median methylation values for each DMR over all cell types were then clustered (_k_-means). Shown are the color-coded methylation values of each DMR (rows), organized into clusters showing higher methylation in fibroblasts than in native stem cells (hyper-DMRs) and clusters showing lower methylation in fibroblasts than in native stem cells (hypo-DMRs). Overall, the clusters reflect different basal levels of methylation across the genome, but good correspondence between methylation in the different ES lines. An important exception is a cluster including DMRs with significantly higher methylation in ORMES-22 than in CRES-2. Some of the DMRs in this cluster may reflect ORMES-22 line-specific effects and were excluded from further analysis. Other DMRs in this cluster are also hypermethylated in the ORMES-9 line and were classified as “failed reprogramming” DMRs and analyzed separately. (B) Distribution of DMR sizes. Shown is the distribution of sizes of genomic intervals determined to be (red) hyper- and (green) hypo-DMRs. Hypo-DMRs have a more specific length distribution, peaking around 2 kb. (C) DMR CpG content. The average number of CpGs in 500-bp windows was computed for each DMR (each CpG was counted twice), and the distribution of CpG contents for hyper- and hypo-DMRs was plotted. Hyper-DMRs have a lower overall CpG content. Importantly, both types of DMRs generally occupy regions of low to medium CpG content, and are not observed in classical CpG islands (CpG content >50).

Figure 3.

Figure 3.

Monkey DMRs are conserved in human ESC and tissues. (A) Muscle-ES differential methylation. Shown are box plots of the DNA methylation differences between human muscle tissues and human ESCs (Straussman et al. 2009), computed for regions of the human genome that are orthologous to monkey (red) hyper- and (green) hypo-DMRs, or to regions with (blue) low or (yellow) high monkey ES methylation. Since the human data span only CpG islands, the statistics only cover regions with intermediate or high CpG content. _P_-values indicate the significance (using KS test) of difference between hyper-DMRs and low ES methylation regions, and between hypo-DMRs and high ES methylation regions. (B) Range of methylation across a panel of human tissues. Shown are box plots for the differences between the minimum and maximum DNA methylation in human brain, colon, spleen, and liver (Irizarry et al. 2009), for regions that are orthologous to monkey DMRs or regions of high and low monkey ES methylation (same color scheme as in A). _P_-values indicate the significance of difference between hypo-DMRs and high ES methylation (for CpG range 0–15) and between hyper-DMRs and low ES methylation (for CpG range 15–40).

Figure 4.

Figure 4.

CTCF and Polycomb are correlated with differential methylation. (A) SUZ12 occupancy. Shown are box plots for average human SUZ12 occupancy on mapped monkey DMRs and background regions. We separately plot groups of regions with different levels of CpG content, dissected into (red) hyper-DMRs, (green) hypo-DMRs, (blue) regions with low ES methylation, and (yellow) regions with high ES methylation. In general, regions with high methylation have low SUZ12 levels (e.g., lower than regions with low methylation; see CpG content 15–40). Moreover, regions with higher CpG content (>40) that are hyper- and hypomethylated are enriched in SUZ12 targets. (B) CTCF occupancy at CTCF motifs. Shown are distributions of CTCF binding levels in three groups of genomic loci: (1, gray) background regions lacking CTCF motifs and having low DNA methylation; (2, red) regions of high methylation featuring CTCF binding motifs; (3, green) regions with low DNA methylation featuring CTCF binding motifs. (C) CTCF binding capacity at DMRs. Shown are cumulative probability distributions for the predicted binding energy of the CTCF motif in (red) hyper-DMRs and (green) hypo-DMRs. About 15% of the hypo-DMRs have a strong CTCF binding site, much higher than the percentage for hyper-DMRs. (D) Combinatorial analysis. Shown are counts of DMRs associated with combinations of regulated TSS, SUZ12 hotspot, or CTCF binding site. More than half of the DMRs have at least one factor associated with them.

Figure 5.

Figure 5.

Partial and failed reprogramming. (A) Failure to reprogram DMRs. Shown are examples of DMRs in which the reprogrammed ES DNA methylation pattern follows the fibroblast pattern. These stand in marked contrast to the overall genomic trend (e.g., Fig. 1) and may represent complete lack of reprogramming, partial reprogramming that could not complete, or ongoing reprogramming with much slower kinetics than the genomic trend. (B) Reprogramming ratios. Reprogramming ratios were computed as the ratio of the difference between the reprogrammed ES and fibroblast methylation medians and the difference between the native ES and fibroblast methylation medians. A ratio of 1 indicates perfect reprogramming, and a ratio of 0 represents no reprogramming. Plotted is the distribution of reprogramming ratios of hypo-DMRs and hyper-DMRs. Data are only shown for DMRs that had similar methylation levels in the two native ES lines (ORMES-22 and ORMES-9).

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