Cyclic DNA remethylation following active demethylation at euchromatic regions in mouse embryonic stem cells
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ORIGINAL ARTICLE
Cyclic DNA remethylation following active demethylation at euchromatic regions in mouse embryonic stem cells Musashi Kubiura-Ichimaru & Takamasa Ito & Louis Lefebvre & Masako Tada
Received: 3 August 2020 / Revised: 8 November 2020 / Accepted: 9 November 2020 # Springer Nature B.V. 2020
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 5hydroxymethylation (5hmC), converted from 5mC by TET-family enzymes, by means of immunofluorescence staining of mitotic chromosomes in mouse Responsible Editor: Tatsuo Fukagawa M. Kubiura-Ichimaru : T. Ito : M. Tada (*) Stem Cells & Reprogramming Laboratory, Department of Biology, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan e-mail: [email protected] L. Lefebvre Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Institute, University of British Columbia, Vancouver V6T 1Z3, Canada
embryonic stem cells (ESCs). Our comparison of immunostaining patterns for those epigenetic modifications in wild-type, DNMT-deficient, and TETdeficient ESCs allowed us to visualise cell cyclemediated 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. Keywords DNA methylation . 5hmC . mouse ESCs . chromatin . cell cycle . immunofluorescence
Background The nucleosome is the primary unit of chromatin and essentially consists of ~ 146 bp of DNA wrapped around two copies of each of the canonical histones H2A, H2B, H3, and H4. Nucleosomes render genomic DNA inert by preventing various regulatory proteins from accessing the DNA (Workman and Kingston 1998; Richmond and Davey 2003). Extensive remodelling is required to produce the nucleosome-free regions (NFRs) that are necessary for DNA replication, repair, and transcription, as well as replication-coupled or replication-independent DNA modification (Felle et al. 2011; Brogaard et al. 2012). In mammalian genomes, 70–80% of all CpG motifs are methylated in somatic cells (Lister et al. 2009). Therefore, most of the g
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