Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase

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.

Editing DNA Methylation in Mammalian Embryos

“…We also applied this technique to embryonic stem (ES) cells lacking all three types of Dnmt genes, Dnmt1, Dnmt3a, and Dnmt3b, called Dnmt triple KO (TKO) ES cells [75], and successful induction of DNA methylation was clearly observed in nuclei with mCherry-MBD-NLS. Similar results have been obtained using dCas9-SssI with gRNA expression in mouse embryos [25]. As an aid to analyzing the biological functions of germ-cell-specific hypomethylation in centromeres and pericentromeres, we are currently conducting research on early preimplantation embryos and germ cells with this technique.…”

“…DNMT3A consists of a regulatory region containing the N-terminal PWWD domain, ADD domain, and a C-terminal catalytic domain. Because it is a large molecule (912 amino acids, aa), only the catalytic domain is used frequently [17], and ZF [18][19][20][21][22][23], TALE [24][25][26], and dCas9 [25,[27][28][29][30][31][32] have been fused with the catalytic domain of DNMT3A. There have been reports on the optogenetic regulation of enzymatic activity of epigenome-editing enzymes.…”

“…ZF-MIWI2 targeted to Line1 retrotransposon induces DNA methylation in male germ cells [23] (Table 1). While mammalian DNA methyltransferase is frequently used as the effector protein for epigenome-editing enzymes, the bacteria-derived methyltransferase M.SssI has also been used as an effector protein for introducing DNA methylation [25,29,30] (Table 1 and Figure 1). Compared with DNMT3A, SssI is a small molecule (386 aa) present in Spiroplasma spp.…”

“…Additionally, the functions of the amino acid residues of SssI have been well studied [38,39]. Because SssI is a strong DNA methyltransferase, it helps to regulate enzymatic activity by controlling the expression of genes [25], using enzymatically optimized SssI mutants [29] or enzyme splitting [30] to decrease off-target effects. There are some advantages to the use of SssI as an effector protein of epigenome-modifying enzymes.…”

“…We previously reported the introduction of DNA methylation into the ncDNA of pericentromeres in mouse embryos [25]. Generally, mouse oocytes are hypomethylated in centromeres and pericentromeres, as described above.…”