Charting a dynamic DNA methylation landscape of the human genome.

Identification and characteristics of differentially methylated regions (DMRs) in the human genomea. Principal component analysis based on CpG methylation levels for 1kb tiles across 30 diverse human cell and tissue samples. Coloring indicates classification of samples into subgroups and group wise mean DNAme. Detailed sample annotations are listed in Supplementary Table 1. Gray area indicates Alzheimer’s disease (AD) samples..b. Density scatterplot of CpG wise DNAme level differences (x-axis, p≤0.01) and CpG median methylation (y-axis) across the 24 developmental samples (excluding cancer and long-term culture). Coloring indicates CpG density from low (blue) to high (red). The red box highlights dynamic CpGs (≥0.3).c. Cumulative distribution of DMR specificity. High hypo/hypermethylation specificity indicates that particular region is methylated/unmethylated in most tissues and deviates from this default state in only one or few cases.d. Top: Composite plot of mean DNAme differences across various genomic features. Black line indicates the median of the average DNAme difference across each feature. Grey areas mark 25th and 75th percentile. Bottom: Distribution of mean DNAme difference for each genomic feature. Black bar indicates 25th and 75th percentile while white dot marks the median. For CGI islands, a smaller, experimentally determined set (eCGI; n=25,490) is shown as well. Promoters are broken down into high CpG content (HCP, n=24,899), intermediate CpG content (ICP, n=10,920) and low CpG content (LCP, n=7,946) regions (n=43,765 total).e. Methylation level variation across the OCT4 locus (chr6:31,119,000–31,162,000) (top). Blue boxes indicate DMRs significant at p≤0.01 and exhibit a minimum difference ≥0.3 across the 24 developmental samples. For reference, ENCODE TFBS cluster track, DNAse I hypersensitive sites, CpG islands and RefSeq genes are shown.f. Distribution of DMRs across various genomic features. Each region is assigned only to one of these genomic feature according the ranking promoter, CGI, CGI shore, exon, intron, putative enhancers, DNAse I hypersensitive site or other.

Dynamic CpG methylation regions frequently co-localize with transcription factor binding sites (TFBS)a. Overlap of DMRs with ENCODE TFBS.b. Enrichment of the top four TFBSs significantly overrepresented (p<0.01, empirical test) in DMRs specific to the cell type indicated (specificity >0.15). Color code quantifies median enrichment odds ratio compared to size matched random control regions.c. Overlap of PAX5 motifs (±100bp top) unmethylated in CD34 cells or fetal brain across the entire human genome. Regions specifically unmethylated in CD34 or fetal brain were subjected to motif analysis and top differentially co-occurring motifs are highlighted on the left for CD34 and on the right for fetal brain.d. Density scatterplot of maximum DNAme difference across 24 developmental samples for TFBS cluster track (n=2.7 million) and median methylation level across all samples. Color code indicates density of TFBS from low (blue) to high (red)..

DMRs exhibit elevated SNP frequency and show non-random GWAS SNP enrichmenta. Odds ratio of significantly overrepresented (p<0.05, empirical test, see Supplementary Information) GWAS SNPs grouped into 16 categories in regions specifically hypomethylated within the sample indicated on the left. Asterisk indicates p-value <0.1.

Effective classification and sample deconvolution using only the DMR seta. Overlap of dynamic CpGs (p≤0.01 Δ≥0.3) in normal samples and between colon cancer and matching control CpG numbers (in million).b. Distribution of autosomal CpGs across three conditions. Class name indicates sample group where a CpG was observed dynamic (developmental (n=24), cell culture (n=3), cancer (n=2)) or remained unchanged over the entire sample set (n=30).c. Repeat content distribution of DMRs (sets as in b).d. Hierarchical clustering using pearson correlation coefficient (PCC) of the DMR values across the entire sample set (n=30).e. Distance of the fetal brain sample to different sets of signature regions defined for sample classes or individual samples, but excluding regions identified by means of the fetal brain sample.f. Contribution of individual sample signature region sets to an in silico generated hybrid sample (HUES64 and hippocampus).