DNMT1 and DNMT3b cooperate to silence genes in human cancer cells

Abstract

Inactivation of tumour suppressor genes is central to the development of all common forms of human cancer1. This inactivation often results from epigenetic silencing associated with hypermethylation rather than intragenic mutations2,3,4,5,6,7. In human cells, the mechanisms underlying locus-specific or global methylation patterns remain unclear8,9. The prototypic DNA methyltransferase, Dnmt1, accounts for most methylation in mouse cells10,11, but human cancer cells lacking DNMT1 retain significant genomic methylation and associated gene silencing12. We disrupted the human DNMT3b gene in a colorectal cancer cell line. This deletion reduced global DNA methylation by less than 3%. Surprisingly, however, genetic disruption of both DNMT1 and DNMT3b nearly eliminated methyltransferase activity, and reduced genomic DNA methylation by greater than 95%. These marked changes resulted in demethylation of repeated sequences, loss of insulin-like growth factor II (IGF2) imprinting, abrogation of silencing of the tumour suppressor gene p16INK4a, and growth suppression. Here we demonstrate that two enzymes cooperatively maintain DNA methylation and gene silencing in human cancer cells, and provide compelling evidence that such methylation is essential for optimal neoplastic proliferation.

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Figure 1: Human cells lacking DNMT3b and DNMT1.
Figure 2: DNMT3b-/-;DNMT1-/- cells are demethylated.
Figure 3: Repetitive regions are demethylated in DNMT3b-/-;DNMT1-/- cells.
Figure 4: Gene silencing in DNMT3b-/-;DNMT1-/- cells.

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Acknowledgements

We thank S. R. Lee and S. G. Rhee for assistance with the HPLC analysis. This work was supported by the Clayton Fund, the V Foundation, and by grants from the National Institutes of Health.

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Correspondence to Stephen B. Baylin or Bert Vogelstein.

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Competing interests

S.B.B. is consultant to Tibotec-Virco. Under licensing agreement between the Johns Hopkins University and Tibotec-Virco, M.S.P. was licensed to Tibotec-Virco and S.B.B. is entitled to a share of the royalties received by the University from sales of the licensed technology. The terms of these arrangements are being managed by the University in accordance with its conflict of interest policies.

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