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.
Highlights
Methylated cytosines in CpG dinucleotides (5mC) play crucial roles in various biological phenomena through regulating gene expression, and aberrant DNA methylation leads to diseases and developmental defects [1]
We designed fusion proteins composed of a TALE recognizing major satellites [30] and enzymatically active (WT) or inactive (T313D) SssI CpG methyltransferase (Fig 1A and 1B)
DAPI-dense signals were well stained with anti-FLAG antibody, and CENPC signals were localized around these heterochromatin foci (Fig 1C)
Summary
Methylated cytosines in CpG dinucleotides (5mC) play crucial roles in various biological phenomena through regulating gene expression, and aberrant DNA methylation leads to diseases and developmental defects [1]. DNA methylation in mouse preimplantation embryos Because TALMaj-SssI had some off-targets toward the Line retrotransposon in our ChIPqPCR analysis (Fig 1E), we used bisulfite sequencing to evaluate the DNA methylation of the major and minor satellites under the dose-dependent expression of TALMaj-SssI in mouse preimplantation embryos that were known to have hypomethylated centromeres and pericentromeres based on previous analysis [10]. In 2-cell stage embryos, MBD signals accumulating at pericentromeric heterochromatin were slightly higher in embryos expressing WT dCas9-SssI compared with the T313D and gRNA combination (Fig 5A). Line DNA methylation was higher than that of TALMaj-SssI expressed embryos (Figs 2B and 6B) As these results demonstrate, fewer off-target effects were shown with the TALE-based system compared with CRISPR/Cas when SssI methyltransferase was used as the effector domain of epigenome editing enzymes
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