Nucleic Acids Research Advance Access originally published online on December 6, 2006
Nucleic Acids Research 2007 35(1):100-112; doi:10.1093/nar/gkl1035
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Nucleic Acids Research, 2007, Vol. 35, No. 1 100-112
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes
1 Institut für Biochemie, FB 08, Heinrich-Buff-Ring 58, Justus-Liebig-Universität Giessen 35392 Giessen, Germany 2 Medical Research Council, Laboratory of Molecular Biology, Structural Studies Division Hills Road, Cambridge CB2 2QH, UK 3 International University Bremen, School of Engineering and Science Campus Ring 1, 28759 Bremen, Germany
*To whom correspondence should be addressed. Tel: +49 421 200 3247; Fax: +49 421 200 3249; Email: a.jeltsch{at}iu-bremen.de
Received July 17, 2006. Revised September 25, 2006. Accepted November 7, 2006.
Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.
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