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J. Biol. Chem., Vol. 283, Issue 15, 9878-9885, April 11, 2008

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Heritable Gene Repression through the Action of a Directed DNA Methyltransferase at a Chromosomal Locus^*

Alexander E. Smith, Paul J. Hurd, Andrew J. Bannister, Tony Kouzarides, and Kevin G. Ford¹

From the Department of Haematological Medicine, The Rayne Institute, King's College London, Guy's, King's, and St. Thomas' School of Medicine, 123 Coldharbour Lane, London SE5 9NU, United Kingdom and the Wellcome Trust and Cancer Research UK Gurdon Institute of Cancer & Developmental Biology and the Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, United Kingdom

The ability to exogenously impose targeted epigenetic changes in the genome represents an attractive route for the simulation of genomic de novo epigenetic events characteristic of some diseases and for the study of their downstream effects and also provides a potential therapeutic approach for the heritable repression of selected genes. Here we demonstrate for the first time the ability of zinc finger peptides to deliver DNA cytosine methylation in vivo to a genomic integrated target promoter when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular genomic de novo methylation events and allowing a direct analysis of the mechanics of de novo DNA methylation-mediated gene silencing at a genomic locus. We show that targeted methylation leads to gene silencing via the initiation of a repressive chromatin signature at the targeted genomic locus. This repression is maintained after the loss of targeted methyltransferase enzyme from the cell, confirming epigenetic maintenance purely through the action of cellular enzymes. The inherited DNA methylation pattern is restricted only to targeted sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. As well as demonstrating the potential of these enzymes as tools for the exogenous, heritable control of cellular gene expression, this work also provides the most definitive confirmation to date for a transcriptionally repressive role for de novo DNA methylation in the cell and lends some weight to the hypothesis that the aberrant methylation associated with certain diseases may well be a cause rather than a consequence of transcriptional gene repression.

Received for publication, December 20, 2007 , and in revised form, February 11, 2008.

^* This work was supported by Grant 47/GTH12521 from the Biotechnology and Biological Sciences Research Council and Grant 06089 from the Leukaemia Research Fund, United Kingdom. Work in the Kouzarides laboratory was supported by Cancer Research UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed. Tel.: 207-848-5909; Fax: 207-733-3877; E-mail: kevin.ford{at}kcl.ac.uk.

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