Abstract
DNA methylation is an essential epigenetic mark that regulates normal mammalian embryonic development. DNA methylation profiles are not always static, especially during germline development. In zygotes, DNA is typically highly methylated but, during preimplantation, DNA methylation is erased globally. Then, at the start of post-implantation development in mouse embryos, DNA again becomes dramatically hypermethylated. Chromatin structure regulates the accessibility of DNA-modifying enzymes to target DNA. Beyond that, however, our understanding of the pathway by which chromatin regulation initiates changes in global DNA methylation during mouse embryonic development remains incomplete. To analyse the relationship between global regulation of DNA methylation and chromatin status, we examined 5-methylcytosine (5mC), modified by the DNA methyltransferase DNMT, and the oxidative derivative 5-hydroxymethylation (5hmC), converted from 5mC by TET-family enzymes, by means of immunofluorescence staining of mitotic chromosomes in mouse embryonic stem cells (ESCs). Our comparison of immunostaining patterns for those epigenetic modifications in wild-type, DNMT-deficient, and TET-deficient ESCs allowed us to visualise cell cycle-mediated DNA methylation changes, especially in euchromatic regions. Our findings suggest that DNA methylation patterns in undifferentiated mouse ESCs are stochastically balanced by the opposing effects of two activities: demethylation by TET and subsequent remethylation by DNMT.
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Abbreviations
- ESCs:
-
mouse embryonic stem cells
- MEFs:
-
mouse embryonic fibroblasts
- WT:
-
wild type
- TKO:
-
triple knockout
- DKO:
-
double knockout
- 5mC:
-
5-methylcytosine
- 5hmC:
-
5-hydroxymethylation
- HtC:
-
heterochromatin
- EuC:
-
euchromatin
- IF:
-
immunofluorescence
- dsDNA:
-
double-stranded DNA
- TSA:
-
trichostatin A
- VPA:
-
valproic acid
- c:
-
unmethylated single-stranded DNA
- m:
-
methylated single-stranded DNA
- h:
-
hydroxymethylated single-stranded DNA
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Acknowledgements
DKO and dTKO ESCs, tTKO ESCs, and anti-H3K9me3 antibody were gifts from Dr. Masaki Okano, Dr. Izuho Hatada, and Dr. Hiroshi Kimura, respectively.
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This work was supported by funds from the JSPS to MT (Grant 24613004) and MK (DC1, 2610904), as well as from the CIHR (PJT-165992) to LL.
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MT conceived of the study conception and design. Data collection and analysis were performed by MK, MT, and TI. LL performed data analysis. MT wrote the first draft of the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Kubiura-Ichimaru, M., Ito, T., Lefebvre, L. et al. Cyclic DNA remethylation following active demethylation at euchromatic regions in mouse embryonic stem cells. Chromosome Res 29, 145–157 (2021). https://doi.org/10.1007/s10577-020-09645-y
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DOI: https://doi.org/10.1007/s10577-020-09645-y