title: Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.
abstract: DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.
authors.affiliation: Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Stepper
authors.affiliation: Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany.
authors.forename: Goran
authors.initials: G
authors.lastname: Kungulovski
authors.affiliation: Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany.
authors.forename: Renata Z
authors.initials: RZ
authors.lastname: Jurkowska
authors.affiliation: Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK; The Welcome Trust Sanger Institute, Cambridge CB10 1SA, UK.
authors.forename: Tamir
authors.initials: T
authors.lastname: Chandra
authors.affiliation: The Babraham Institute, Cambridge CB22 3AT, UK.
authors.forename: Felix
authors.initials: F
authors.lastname: Krueger
authors.affiliation: Max Planck Genome-Centre Cologne, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.affiliation: Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK; The Welcome Trust Sanger Institute, Cambridge CB10 1SA, UK.
authors.forename: Wolf
authors.initials: W
authors.lastname: Reik
authors.affiliation: Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
authors.affiliation: Institute of Biochemistry, Pfaffenwaldring 55, Faculty of Chemistry, University of Stuttgart, D-70569 Stuttgart, Germany.
authors.forename: Tomasz P
authors.initials: TP
authors.lastname: Jurkowski
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D018899:CpG Islands; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D004720:Endonucleases