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. 2012 May;22(5):837-49.
doi: 10.1101/gr.131169.111. Epub 2012 Mar 5.

A DNA hypermethylation module for the stem/progenitor cell signature of cancer

Hariharan Easwaran  1 , Sarah E Johnstone, Leander Van Neste, Joyce Ohm, Tim Mosbruger, Qiuju Wang, Martin J Aryee, Patrick Joyce, Nita Ahuja, Dan Weisenberger, Eric Collisson, Jingchun Zhu, Srinivasan Yegnasubramanian, William Matsui, Stephen B Baylin
Affiliations

A DNA hypermethylation module for the stem/progenitor cell signature of cancer

Hariharan Easwaran et al. Genome Res. 2012 May.

Abstract

Many DNA-hypermethylated cancer genes are occupied by the Polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that ~75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESCs and adult stem/progenitor cells. A large number of these genes are key developmental regulators, and a subset, which we call the "DNA hypermethylation module," comprises a portion of the PcG target genes that are down-regulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find that these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions.

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Figures

Figure 1.
Figure 1.
Genes with promoter-proximal CpG hypermethylation in osteosarcoma are greatly enriched for a bivalent chromatin history in ESCs and are down-regulated in osteosarcoma cells compared with ESCs. (A) Schematic of the experimental design. von Kossa staining shows differentiation of MSCs to osteoblasts (scale bar, 50 μm). (B) Heat map of β-values for 2489 Infinium probes (Supplemental Table 1) within CpG islands of 1891 genes for cell lines corresponding to different tumor types. These are probes that are methylated in at least one cell line and not methylated in any of the normal cells (see Methods). Different cell types are shown below the plot. (C) Heat map of the β-values for MSCs, osteoblasts, and U2OS cells. Genes with β-value >0.75 in U2OS and <0.25 in MSCs and osteoblasts were selected as hypermethylated genes in osteosarcoma. (D) Extent of overlap of the osteosarcoma-hypermethylated genes with genes marked by H3K4Me3 or H3K27Me3 in ESCs. Osteosarcoma-hypermethylated genes overlap significantly with ESC-bivalent genes (P-value < 0.001).
Figure 2.
Figure 2.
Plots of H3K4Me3 and H3K27Me3 enrichment in MSCs (M-K4Me3 and M-K27Me3), osteoblasts (O-K4Me3 and O-K27Me3), U2OS (U-K4Me3 and U-K27Me3), and ESCs (ESC-K4Me3 and ESC-K27Me3). (*) Cell type in which the gene is identified as methylated. (Green and red boxes) Peak height normalized to background enrichment for H3K4Me3 and H3K27Me3, respectively. Genes represented include CDKN2A (p14), CDKN2B (p15), and CDKN2A (p16) (A), DLX5 (B), HIC1 (C), and GPR101 (D).
Figure 3.
Figure 3.
Enrichment of chromatin marks and levels of gene expression in ESCs, MSCs, and osteoblasts for genes methylated in U2OS. (Pie chart) Proportion of genes methylated in U2OS that have the different chromatin marks or none of the marks analyzed in this study in ESCs (A), MSCs (B), or osteoblasts (C). (Lower panels) Array expression intensities (log2) in ESCs, MSCs, or osteoblasts of gene probes constituting these subsets; in each case, they are compared to that in U2OS. (*) Significant gene expression changes (P-value < 0.005). (Gray line in the plot) Median log2 intensity of all genes in the corresponding cell type. See also Supplemental Figures 1 and 3.
Figure 4.
Figure 4.
Genes hypermethylated in a wide range of cancers are enriched for bivalent chromatin in ESCs and HSCs or H3K27Me3 in MSCs and CD36 erythroid progenitor cells or osteoblasts. Plots of the percentage of methylated genes tracing to each chromatin category (indicated by color for the chromatin type) of each of the cell types listed are indicated for each of the tumor types listed on the x-axis. (Dashed lines) The 50% mark for fraction of methylated genes. (A–E) Plots track the histone marks present for methylated genes based on the chromatin composition of ESCs, HSCs, MSCs, CD36, and osteoblasts, respectively. (F) Summary of the chromatin marks in each cell type for genes that are hypermethylated in at least 50% of cell lines in each tumor type.
Figure 5.
Figure 5.
Cancer-specific hypermethylated genes are enriched for functions associated with developmental regulation and comprise a list of genes that are tightly silenced in tumor cells compared with MSCs. Enrichment of biological processes in hypermethylated genes marked by bivalency (A) or H3K4Me3 (B) in ESCs against a background list of genes that, respectively, have the same mark and are present on the methylation array. (Right) Top five molecular function categories of the genes constituting the significantly enriched biological processes. The numbers adjacent to the bars are the total number of genes in the bivalent or H3K4Me3-marked genes in that category (denominator) and the number of genes in the category that are DNA hypermethylated in 50% or more of all the cancers examined (numerator). Supplemental Tables 2 and 3 give the gene lists used for GO analysis and all the top categories identified.
Figure 6.
Figure 6.
PRC-module and ESC-bivalent genes undergo significant repression of expression upon hypermethylation in cancer. (A, top panel) Venn plot of the overlap between the 384 genes hypermethylated in U2OS and the PRC module (Kim et al. 2010) (left) or the ESC-bivalent module (right). (Bottom panel) Expression changes between U2OS and MSCs for the Myc, Core, PRC-modules, ESC-bivalent genes, and the set of genes hypermethylated in osteosarcoma (termed “U2OS Methylome”). (*) Significant change from background based on Wilcoxon rank-sum test. The methylated genes that are not bivalent in ESCs (called “Me no bivalent”) are all marked by H3K4Me3. (B) Effect of 5-deoxy-aza-cytidine (DAC) and trichostatin (TSA) on re-expression of U2OS-hypermethylated genes marked by H3K4Me3 or H3K27Me3 in MSCs (B) or those marked by H3K4Me3, bivalent or PRC in ESCs (C). The log2 gene expression ratios for these genes from Agilent gene expression data summarizing the relative expression of osteoblasts versus MSCs, U2OS versus MSCs, and the relative expression of DAC- or TSA-treated MSCs, osteoblasts, and U2OS versus their control-treated counterparts are shown in B and C. (*) P-values < 0.001. (D) Model depicting the change of repressive mechanism from bivalent/PcG-marking to DNA hypermethylation. Developmental regulator genes are marked by bivalent (embryonic/adult stem cell) or PcG (adult stem/early progenitor cells) and have relatively low levels of expression. In tumors, DNA hypermethylation (mC) of bivalent/PcG-marked genes in stem cells leads to tight silencing at these genes. This epigenetic switch is responsible for the stable silencing seen at developmental regulators in cancer cells and may explain how cancer cells recapitulate aspects of the transcriptional and phenotypic state of stem cells.
Figure 7.
Figure 7.
Hierarchical cluster analysis of primary TCGA colon and breast cancer samples based on Infinium β-values of PRC-module genes identified as methylated in colon (A) or breast (B) cancer cell lines. (A) Colon tumors are classified as CIMP+ (top red bars in the heat map) if eight or more of a 12-CIMP marker panel (Weisenberger et al. 2006) are methylated in a tumor sample, and otherwise classified as CIMP− (top gray bars in the heat map). (Red) Tumor and (green) normal (T/N) samples. (B) Breast tumor samples were classified as estrogen (ER)/progesterone (PR) positive or negative and also classified according to their gene expression (PAM50) classes. The PRC-module genes sufficiently discern the CIMP+ and CIMP− tumors in both colon and breast samples and tightly cluster important breast cancers subtypes that are independently defined based on gene expression signatures.
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