Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes.
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.
Li
Fuyang
F
Institut für Biochemie, FB 08, Heinrich-Buff-Ring 58, Justus-Liebig-Universität Giessen, 35392 Giessen, Germany.
Papworth
Monika
M
Minczuk
Michal
M
Rohde
Christian
C
Zhang
Yingying
Y
Ragozin
Sergei
S
Jeltsch
Albert
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D020868:Gene Silencing; D011905:Genes, ras; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000254:growth & development; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics; D016335:Zinc Fingers; D000097584:DNA Methyltransferase 3B
D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse