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Increased methylation variation in epigenetic domains across cancer types


Tumor heterogeneity is a major barrier to effective cancer diagnosis and treatment. We recently identified cancer-specific differentially DNA-methylated regions (cDMRs) in colon cancer, which also distinguish normal tissue types from each other, suggesting that these cDMRs might be generalized across cancer types. Here we show stochastic methylation variation of the same cDMRs, distinguishing cancer from normal tissue, in colon, lung, breast, thyroid and Wilms' tumors, with intermediate variation in adenomas. Whole-genome bisulfite sequencing shows these variable cDMRs are related to loss of sharply delimited methylation boundaries at CpG islands. Furthermore, we find hypomethylation of discrete blocks encompassing half the genome, with extreme gene expression variability. Genes associated with the cDMRs and large blocks are involved in mitosis and matrix remodeling, respectively. We suggest a model for cancer involving loss of epigenetic stability of well-defined genomic domains that underlies increased methylation variability in cancer that may contribute to tumor heterogeneity.

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Figure 1: Increased methylation variance of common CpG sites across human cancer types.
Figure 2: Large hypomethylated genomic blocks in human colon cancer.
Figure 3: Loss of methylation stability at small DMRs.
Figure 4: Adenomas show intermediate methylation variability.
Figure 5: High variability of gene expression associated with blocks.

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We thank C. Adams and Applied Biosystems, Inc. for supplying reagents for the sequencing experiments, B. Vogelstein, F. Giardiello and M. Zeiger for tumor samples and M. Newhouse for computer assistance. This work was supported by US National Institutes of Health grants R37CA054358, R01HG005220, 5P50HG003233, F32CA138111, 5R01GM083084 and R01DA025779 (K.Z.).

Author information

Authors and Affiliations



K.D.H. and R.A.I. wrote the DMR finder and smoothing algorithms. W.T. performed and analyzed the arrays with H.C.B., who wrote new software for this purpose. S.S. made the libraries and performed validation. B.L. wrote new methylation sequence alignment software. O.G.M. performed the histopathologic analysis. B.W. and H.W. performed LOCK experiments. Y.L. performed copy number experiments. D.D. and K.Z. performed bisulfite capture. E.B. performed the sequencing. R.A.I. and A.P.F. conceived and led the experiments and wrote the paper with the predominant assistance of K.D.H., W.T., H.C.B. and B.L.

Corresponding authors

Correspondence to Rafael A Irizarry or Andrew P Feinberg.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Note, Supplementary Tables 1, 2, 4–6, 8–10 and 13–19 and Supplementary Figures 1–24. (PDF 15293 kb)

Supplementary Table 3

List of block locations (XLS 2912 kb)

Supplementary Table 7

List of small DMRs (XLS 1969 kb)

Supplementary Table 11

List of genes showing statistically significant over-expression in cancer compared to normal samples and are within 2,000 bp from an outward methylation boundary shift. (XLS 263 kb)

Supplementary Table 12

Genes with higher gene expression variability in cancer compared to normal. (XLS 170 kb)

Supplementary Table 20

As Supplementary Table 3, but for sample-specific blocks. (XLS 8811 kb)

Supplementary Table 21

As Supplementary Table 7, but for sample-specific small DMRs. (XLS 5053 kb)

Supplementary Table 22

Primers used for bisulfite pyrosequencing (XLS 35 kb)

Supplementary Table 23

List of microarrays used to identify tissue-specific genes (XLS 87 kb)

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Hansen, K., Timp, W., Bravo, H. et al. Increased methylation variation in epigenetic domains across cancer types. Nat Genet 43, 768–775 (2011).

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