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Molecular and Cellular Biology, October 2004, p. 8862-8871, Vol. 24, No. 20
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.20.8862-8871.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Severe Global DNA Hypomethylation Blocks Differentiation and Induces Histone Hyperacetylation in Embryonic Stem Cells

Melany Jackson,1 Anna Krassowska,1 Nick Gilbert,2 Timothy Chevassut,1 Lesley Forrester,1 John Ansell,1 and Bernard Ramsahoye1*

John Hughes Bennett Laboratory, Division of Oncology, School of Molecular and Clinical Medicine, University of Edinburgh,1 Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom2

Received 13 February 2004/ Returned for modification 26 April 2004/ Accepted 18 June 2004

It has been reported that DNA methyltransferase 1-deficient (Dnmt1–/–) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a–/–,3b–/–] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a–/–,3b–/–] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a–/–,3b–/–] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1–/– ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1.

* Corresponding author. Mailing address: John Hughes Bennett Laboratory, Division of Oncology, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom. Phone: 44 131 537 1763. Fax: 44 131 467 8450. E-mail: Bernard.Ramsahoye{at}ed.ac.uk.

Molecular and Cellular Biology, October 2004, p. 8862-8871, Vol. 24, No. 20
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.20.8862-8871.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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