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The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores

Abstract

For the past 25 years, it has been known that alterations in DNA methylation (DNAm) occur in cancer, including hypomethylation of oncogenes and hypermethylation of tumor suppressor genes. However, most studies of cancer methylation have assumed that functionally important DNAm will occur in promoters, and that most DNAm changes in cancer occur in CpG islands. Here we show that most methylation alterations in colon cancer occur not in promoters, and also not in CpG islands, but in sequences up to 2 kb distant, which we term 'CpG island shores'. CpG island shore methylation was strongly related to gene expression, and it was highly conserved in mouse, discriminating tissue types regardless of species of origin. There was a notable overlap (45–65%) of the locations of colon cancer–related methylation changes with those that distinguished normal tissues, with hypermethylation enriched closer to the associated CpG islands, and hypomethylation enriched further from the associated CpG island and resembling that of noncolon normal tissues. Thus, methylation changes in cancer are at sites that vary normally in tissue differentiation, consistent with the epigenetic progenitor model of cancer, which proposes that epigenetic alterations affecting tissue-specific differentiation are the predominant mechanism by which epigenetic changes cause cancer.

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Figure 1: Most tissue-specific differential DNA methylation is located at CpG island shores.
Figure 2: Distribution of distance of T-DMRs and C-DMRs from CpG islands.
Figure 3: Similar numbers of sites of hypomethylation and hypermethylation in colon cancer.
Figure 4: Gene expression is strongly correlated with T-DMRs at CpG island shores.
Figure 5: Genes downregulated in association with T-DMR shore hypermethylation are activated by 5-aza-2′-deoxycytidine treatment of colon cancer cell line HCT116 and knockout of DNA methyltrasferase 1 and 3b in HCT116.
Figure 6: Clustering of human tissue samples using mouse T-DMRs results in perfect discrimination of tissues.
Figure 7: Clustering of normal tissue samples using C-DMRs results in perfect discrimination of tissues.
Figure 8: Magnitude of differential methylation and variation in C-DMRs and T-DMRs.

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Acknowledgements

We thank B. Volgelstein (Johns Hopkins University School of Medicine) for providing colon tumors and matched normal mucosa samples. Postmortem brain, liver and spleen tissue was donated by The Stanley Medical Research Institute collection courtesy of M.B. Knable, E.F. Torrey and R.H. Yolken, whom we also thank for making available gene expression data for the brain and the liver tissue. We thank B. Carvalho for help with statistical software and C. Crainiceanu for advice with statistical methods. This work was supported by US National Institutes of Health grants P50HG003233 (A.P.F.), R37CA54358 (A.P.F.) and 5R01RR021967 (R.A.I.).

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Authors and Affiliations

Authors

Contributions

R.A.I. and A.P.F. designed the study and interpreted the results; R.A.I. designed new CHARM arrays and statistical methods with Z.W.; C.L.-A. performed bisulfite pyrosequencing, real-time quantitative PCR and sample preparation with C.M., K.G., M.R. and H.J.; B.W. and S.S. performed CHARM assays with sample preparation from M.W. and advice from J.B.P.; P.O. and H.C. performed functional assays; A.P.F. supervised the laboratory experiments and wrote the paper with R.A.I. and C.L.-A.

Corresponding authors

Correspondence to Rafael A Irizarry or Andrew P Feinberg.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–7 and Supplementary Methods (PDF 541 kb)

Supplementary Data 1

Excel file of T-DMRs (XLS 3629 kb)

Supplementary Data 2

Excel file of C-DMRs (XLS 696 kb)

Supplementary Data 3

Gene expression data from 5-aza-2′ deoxycytidine/DKO experiments and gene expression data for genes associated with T-DMRs (XLS 40 kb)

Supplementary Data 4

Excel file of C-DMRs that are also T-DMRs; and C-DMRs that distinguish all the tumors from normal (XLS 284 kb)

Supplementary Data 5

Mus musculus T-DMRs (XLS 3273 kb)

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Irizarry, R., Ladd-Acosta, C., Wen, B. et al. The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet 41, 178–186 (2009). https://doi.org/10.1038/ng.298

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