Generation and validation of genome-wide CpG methylation maps of APC^Min mouse normal and adenoma tissues.

<p>a) Summary of tissue samples used for genome-wide analyses. B6 wildtype (B) and isogenic B6-APC^Min (APC^Min)mice were employed for MeDIP-seq (M) and RNA-seq (R) of normal intestinal tissue (B, N) and intestinal adenoma (Ad). b) Visualisation of the adenoma-hypermethylated DMR in <i>Ush1g</i>, using the UCSC browser. Maximal height for visualization was set to rpm = 2 for all MeDIP-seq tracks. Black bars, regions that were validated by SIRPH or bisulfite-pyrosequencing (see below, d, e); green, CpG density; blue, purple, red: MeDIP-seq tracks of B6 mouse normal intestine, APC^Min mouse normal intestine, and APC^Min adenoma, respectively. Mice/samples are numbered consecutively. c) Distribution of DMRs in different subgenomic compartments. Odds ratios (i.e. fraction of experimentally observed DMRs divided by relative size of subgenomic compartment) of hyper- and hypomethylation within CpG islands (CGI), promoters that contain or do not contain CGIs, promoter-to-exon junctions, exons, introns, intergenic and repeat regions are given. Dashed line demarcates over- versus underrepresentation. d)–f) Validation of genome-wide MeDIP-seq data, using bisulfite pyrosequencing methodology d) Validation of DMR within <i>Ush1g</i> by bisulfite pyrosequencing using two samples that were subjected to MeDIP-seq and nine additional samples. Percent Methylation of all CpGs across the complete regions is given, colour code as in b). e) High-resolution graphical reconstruction of bisulfite pyrosequencing results for <i>Ush1g</i> DMR region 1, samples B3, N5, Ad5. Red: Methylated; blue: Unmethylated CpG f) Comparison of MeDIP-seq and bisulfite pyrosequencing data, as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003250#pgen.1003250.s012" target="_blank">Table S4c</a>. y-axis represents MeDIP-seq derived and MEDIPS normalized rms-values (log2 scale) for cross-validated genomic regions from three samples (one sample each B6, APC^Min normal, APC^Min adenoma). Box plots depict MeDIP rms values for different methylation classes, as defined by bisulfite pyrosequencing. It is of note that MeDIP-seq procedures cannot detect DMRs with constant reliability over the complete genome, and may under-represent repetitive regions and regions with low CpG density.</p

A model for stepwise formation of cancer cell CpG epigenomes.

<p>CpG methylation is uniform within the normal cellular hierarchy of the intestine, and PRC2-associated H3K27me3 marks are present in crypt and villus cells (blue, to the left). Upon tumour initiation, recurring CpG methylation patterns form, guided by an instructive mechanism that is linked to PRC2 for hypermethylated sites (blue to green). Further CpG methylation changes occur slowly, probably in a stochastic manner. A fraction of these bestow tumour cells with a selective advantage and are subject to clonal expansion during tumour progression (green to red).</p

A core set of APC^Min adenoma-specific CpG methylation patterns is conserved in human colon cancer.

<p>a) GSEA identifies methylation changes of mouse adenoma in human colon cancer. Gene signatures comprise genes with promoter hypo- or hypermethylation in mouse adenoma (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003250#pgen-1003250-g004" target="_blank">Figure 4a</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003250#pgen.1003250.s013" target="_blank">Table S5</a>), genes were ordered by directional methylation changes in human colon cancer (normal tissue versus carcinoma). Mouse and human gene homologues were matched using ENSEMBL Biomart (approx. 14300 unique orthologue pairs were identified). b) Promoter hypo- and hypermethlyation is conserved between mouse APC^Min adenoma and human colon cancer. Genes were selected from those that are significantly hyper- or hypomethylated in APC^Min adenoma. Conserved genes were identified as the core enrichment group of GSEA analysis in a). Figure shows top eleven hypo- and hypermethylated genes in human colon cancer. blue: low relative methylation; red: high relative methylation.</p

Differential gene methylation and differential gene transcription in adenoma do not correlate extensively.

<p>a)–b) Venn diagrams displaying numbers of transcriptionally regulated and differentially methylated genes. Cut-off criteria were FDR<0.001 for transcriptional regulation, and P<0.01 for methylation (calculation using edgeR). a) Genes that display differential methylation of promoter (−1.0 to +0.5 bk relative to transcription start site) b) Genes that display differential gene body methylation. c) GSEA analyses of gene signatures comprising Wnt targets, ISC-, proliferative- and differentiated cell-specific genes. Left panels: analysis of gene activity, as assessed by RNA-seq; middle and right panels: analysis of promoter or gene body methylation. Data is given as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003250#pgen-1003250-g002" target="_blank">Figure 2a</a>. d) Relative gene expression and promoter methylation data for 31 selected epigenetically regulated tumour suppressor genes. Fold change between normal and adenoma is given. <i>Crabp1</i> and <i>Runx3</i> are both, promoter hypermethylated and transcriptionally down-regulated. Asterisk denotes P<0.05.</p

Hypermethylated DMRs are associated with Polycomb targets.

<p>a) Gene Set Enrichment Analysis (GSEA) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003250#pgen.1003250-Subramanian1" target="_blank">[35]</a> is used to probe established epigenetic signatures. Mouse genes were ordered by normal (6 samples) versus adenoma (5 samples) promoter methylation (−1,0 to +0,5 kb). Gene signatures comprising PRC1/2 target genes or mouse homologues of human targets of PRC2 complexes, EED targets, MLL targets or TET1 targets were mapped onto the ordered list, and enrichment at the extremes (hypo- or hypermethylation) was assessed. PRC and EED targets were found strongly enriched among hypermethylated promoters, while no enrichment was detected for MLL targets. TET1 targets were found weakly enriched among hypermethylated promoters, probably due to their known association with PRC complexes, and prevalence at CpG-rich sites <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003250#pgen.1003250-Williams1" target="_blank">[34]</a>. Enrichment score graphs (top, green), signature gene distributions (black line graphs, below ES curves), p-values and false discovery rates (FDRs) are given. Significance cut-offs were P<0.05, FDR<0.25. b) Analysis of H3K27me3 marks in chromatin of mouse intestinal epithelium (n = 4 biological replicates) and adenoma (n = 3), using chromatin immunoprecipitation, followed by qPCR. black bars: Immunoprecipitated chromatin, grey: Input chromatin. Error bars give standard deviation. c) Expression of genes coding for PRC2 components or DNA methyl transferases, as determined by RNA-seq. Gene expression is colour-coded: red, high relative expression; blue, low relative expression. d) Immunohistochemical staining of EED in mouse intestine and adenoma. Dotted line demarcates normal intestinal tissue from adenoma. Adenoma contains higher levels of cytoplasmic and nuclear EED protein. e) Immunofluorescence analysis of DNMT1 in a section of normal intestine and adjacent adenoma of the mouse. Adenoma displays distinct nuclear fluorescence for DNMT1. Scale bar is 50 µm.</p

CpG methylation differentiates normal epithelial cell types from adenoma.

<p>a) Schematic representation of intestinal tissues and cell types. Differentiated villus cells are the prevailing component of bulk normal tissue samples. b) Colour-coded table of CpG methylation analyses of 11 DMRs, using bisulfite pyrosequencing. <i>Cd133</i> is an intestinal stem cell and cancer stem cell marker. <i>Dusp6</i> to <i>Slc25a28</i> represent adenoma hypomethylated DMRs, <i>Ush1g</i>_1 to <i>Vdr</i> represent adenoma hypermethylated DMRs. Percent CpG methylation within the regions are given as numbers and colour-code. Dark blue, <20% CpG methylation; light blue, 20–50% CpG methylation, light red, 50–80% CpG methylation; bright red, >80% methylation. c) Hierarchical clustering of methylation data (as shown in b) separates adenoma from normal tissue and cell preparations. Pearson correlation was employed.</p