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Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase

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 inv...

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Autores principales: Stepper, Peter, Kungulovski, Goran, Jurkowska, Renata Z., Chandra, Tamir, Krueger, Felix, Reinhardt, Richard, Reik, Wolf, Jeltsch, Albert, Jurkowski, Tomasz P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389507/
https://www.ncbi.nlm.nih.gov/pubmed/27899645
http://dx.doi.org/10.1093/nar/gkw1112
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author Stepper, Peter
Kungulovski, Goran
Jurkowska, Renata Z.
Chandra, Tamir
Krueger, Felix
Reinhardt, Richard
Reik, Wolf
Jeltsch, Albert
Jurkowski, Tomasz P.
author_facet Stepper, Peter
Kungulovski, Goran
Jurkowska, Renata Z.
Chandra, Tamir
Krueger, Felix
Reinhardt, Richard
Reik, Wolf
Jeltsch, Albert
Jurkowski, Tomasz P.
author_sort Stepper, Peter
collection PubMed
description 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.
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spelling pubmed-53895072017-04-24 Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase Stepper, Peter Kungulovski, Goran Jurkowska, Renata Z. Chandra, Tamir Krueger, Felix Reinhardt, Richard Reik, Wolf Jeltsch, Albert Jurkowski, Tomasz P. Nucleic Acids Res Gene regulation, Chromatin and Epigenetics 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. Oxford University Press 2017-02-28 2016-11-28 /pmc/articles/PMC5389507/ /pubmed/27899645 http://dx.doi.org/10.1093/nar/gkw1112 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Gene regulation, Chromatin and Epigenetics
Stepper, Peter
Kungulovski, Goran
Jurkowska, Renata Z.
Chandra, Tamir
Krueger, Felix
Reinhardt, Richard
Reik, Wolf
Jeltsch, Albert
Jurkowski, Tomasz P.
Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase
title Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase
title_full Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase
title_fullStr Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase
title_full_unstemmed Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase
title_short Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase
title_sort efficient targeted dna methylation with chimeric dcas9–dnmt3a–dnmt3l methyltransferase
topic Gene regulation, Chromatin and Epigenetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389507/
https://www.ncbi.nlm.nih.gov/pubmed/27899645
http://dx.doi.org/10.1093/nar/gkw1112
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