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  • purchase KN-93 Previously we succeeded in visualizing the DN

    2018-11-06

    Previously, we succeeded in visualizing the DNA methylation status by injecting a GFP-fused MBD of MBD1 protein (EGFP-MBD-NLS) mRNA into living mice zygotes. Using this method, we visualized the DNA methylation dynamics in preimplantation mouse embryos and found that DNA hypomethylation of major and minor satellites is a key signature that distinguishes germ cells from somatic cells (Yamagata et al., 2007; Yamazaki et al., 2007). Importantly, this method cannot only track epigenetic changes in four dimension, but can also achieve it at the single-cell resolution, which was difficult using the conventional methods. However, because mRNA injection was used to express the probe, these epigenetic live-cell imaging studies were limited to preimplantation-stage embryos (until morula stage), and advanced stages of embryonic and fetal development as well as organismal-level analyses were not possible. Mouse embryonic stem cells (ESCs) were originally derived from the inner cell mass of the purchase KN-93 (Evans and Kaufman, 1981), and these cells are developmentally advanced compared to early cleavage- and morula-stage embryos. This was indicated by the fact that trophoblast cells seldom arise from ESCs (Beddington and Robertson, 1989), even after they were cultured in chemical inhibitors “2i”-containing media to maintain naive pluripotency (Morgani et al., 2013). These observations implicate that irreversible epigenetic conversion has taken place in ESCs, whereas it was derived from early embryonic cells. Indeed, although recent studies using high-throughput DNA-sequencing analyses, either combined with bisulfite sequencing or chromatin immunoprecipitation (ChIP), have uncovered the epigenetic differences between preimplantation embryos and ESCs (Habibi et al., 2013; Smith et al., 2012; Yamaji et al., 2013), when these epigenetic changes occur and how these are reflected to 3D chromatin structures are still not known. Here, to track the dynamic changes in this major repressive epigenetic marker during development and differentiation, especially in the ESC-derivation process, we have knocked in a red fluorescent protein (RFP)-fused MBD reporter probe (mCherry-MBD-NLS) into the ROSA26 locus and generated a mouse strain that captures global DNA methylation status in living conditions. Using this reporter mouse, we have discovered that heterochromatin structure, which contains hypermethylated DNA, was highly dynamic in preimplantation embryo, whereas this dynamics was greatly reduced in pluripotent ESCs. We also found that this heterochromatin fixation occurred during the ESC-derivation process, revealed by live-cell imaging analyses. Thus, this model will become a powerful bioresource and technique for understanding DNA methylation dynamics in developmental biology, stem cell biology, and in disease states.
    Results
    Discussion Accumulating evidence indicates that DNA methylation changes dynamically during development, differentiation, pathological processes, and in response to environmental cues (Bergman and Cedar, 2013; Feil and Fraga, 2011; Reik, 2007; Waterland and Jirtle, 2004). Hence, there is a strong need for new experimental approaches to study DNA methylation dynamics sequentially and quantitatively at the organismal level. In this report, we have shown clearly that the MethylRO mouse is viable and fertile and can be used to visualize DNA methylation patterns without any fixation or treatments. Consistent with previous reports by Kobayakawa et al. (2007), Tsumura et al. (2006), and Yamazaki et al. (2007), we have provided multiple evidences that the majority of the mCherry-MBD-NLS probe was concentrated at heterochromatic regions, especially at pericentromeric heterochromatin in preimplantation embryonic cells, ESCs, and somatic cells. Intriguingly, we have discovered that the heterochromatin of preimplantation embryos is highly dynamic because it did not stay at one certain position; rather, it changed its location dramatically in interphase nuclei. In addition, heterochromatic foci not only moved around inside nuclei but also disappeared depending on the cell-cycle status; in contrast, these foci never disappeared in ESCs, indicating that heterochromatin is already fixed, and this fixation actually occurred during the ESC-derivation process. Thus, heterochromatin fixation could be the reason for the difficulty in reversing ESCs to totipotent embryonic cell state (Beddington and Robertson, 1989; Morgani et al., 2013). Moreover, we succeeded in capturing the dynamic changes in DNA methylation status and its pattern in each cell lineage during the ESC-derivation process. Accordingly, these data suggest that the heterochromatin dynamics and stability can be markers of the cellular differentiation status, and further support the idea that chromatin plasticity decreases upon differentiation (Meshorer et al., 2006). Importantly, although we found that H2B signals can be used to calculate the heterochromatin indexes, the heterochromatin pattern was not so clear in visual as compared to MBD probe signals (Figures S3G and S3H). Therefore, it is not suitable to normalize MBD probe signal against that of H2B because this will cancel out the differences seen in the MBD (Figure S3I). Although the MBD reporter was designed to report DNA methylation, this result (the correlation with H2B-EGFP) suggests that the difference in MBD signals for some cell types may reflect a change in heterochromatin organization, and not necessarily in DNA methylation. On the other hand, this also calls for attention toward current conventional studies using immunohistochemistry because fixed cells do not provide information regarding the cell-cycle status or the differentiation process in single cells. Hence, MethylRO mice will be useful for the study of the nuclear dynamics of heterochromatin during proliferation, differentiation, and development.