RRC ID 48844
著者 Yamazaki T, Hatano Y, Handa T, Kato S, Hoida K, Yamamura R, Fukuyama T, Uematsu T, Kobayashi N, Kimura H, Yamagata K.
タイトル Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase.
ジャーナル PLoS One
Abstract To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10-20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50-60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.
巻・号 12(5)
ページ e0177764
公開日 2017-5-18
DOI 10.1371/journal.pone.0177764
PII PONE-D-16-50170
PMID 28542388
PMC PMC5436701
MeSH Animals Base Sequence Cell Line Centromere / genetics* Clustered Regularly Interspaced Short Palindromic Repeats / genetics DNA (Cytosine-5-)-Methyltransferases / metabolism* DNA Methylation / genetics* DNA, Satellite / genetics Embryo Implantation Gene Editing / methods* Genomics* Mice Up-Regulation / genetics
IF 2.74
引用数 10
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
ヒト・動物細胞 Dnmt1-/-Dnmt3a-/-Dnmt3b-/- ES (clone19)(AES0146)