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  • Here we showed that microinjection of siRNA at the

    2021-10-09

    Here we showed that microinjection of siRNA at the MII stage efficiently reduced endogenous EZH2 mRNA and protein. Although the percentage of cleaving embryos was not affected, Alamethicin australia formation and blastocyst cell number were both reduced in EZH2 knockdown groups (Table 1). Similar results have been reported in mouse after loss of Ezh2 at the pronuclear stage [40]. Ezh2-deficient blastocysts have compromised potential for outgrowth, preventing the establishment of ES cell and trophoblast cell lineages [41]. Furthermore, the mutation or maternal depletion of Ezh2 in mouse embryos results in failures in post-implantation development [12,35]. The above studies support the notion that EZH2 plays an important role in embryonic development. EZH2 is a lysine methyltransferase for H3K27me3. Our results showed that EZH2 knockdown moderately decreased the levels of H3K27me3 at the 4-cell and the blastocyst stages. In contrast, it temperately increased the levels of H3K4me3 and reduced the abundance of H3K4me3 histone demethylase KDM5B at both 4-cell and blastocyst stages. Previous research has identified bivalent modifications of H3K27me3 and H3K4me3 in KDM5B-depleted porcine embryos and ESCs [23,37]. The bivalent domain is associated with genes poised for activation during embryonic development, e.g. early differentiation-related genes GATA3, GATA6 and HAND1, which are all increased as a result of EZH2 knockdown (Fig. 5F) [42]. Gata3 is a trophoblast-specific gene that is associated with suppression of pluripotency [43,44]. There is evidence that ectopic expression of PRC2 in mTSCs induces deposition of the H3K27me3 mark at the Gata3 locus and abrogates its transcription. Furthermore, embryos with depleted Kdm5b or ectopic expression of Eed contain abrogated expression of Gata3 in the TE lineage, resulting in them either failing to develop to the blastocyst, or failing to implant [45,46]. HAND1 has been reported to be transcriptionally repressed by PRC2 through bivalent chromatin modifications in both human and murine ES cells [10,23]. The expression of HAND1 was increased in EZH2-deficient porcine parthenotes but not significantly. This is similar to that in mouse embryos, indicating that there are other alternate transcription factors affecting cell fate together [40]. In the present study, the abundance of three pivotal stemness genes, OCT4, SOX2 and NANOG, were also affected at the blastocysts stage after EZH2 knockdown. Consistently, inhibition or knockdown of Ezh2 impairs the reprogramming process in cloned mouse embryos, ESCs, or iPSCs, mainly because of aberrant expression of development-related genes and unscheduled differentiation resulting from the lack of PRC2-dependent H3K27me3 [10,17,47]. A previous study found that Ezh2 co-localizes with development-related genes in the ICM of mouse blastocysts [34]. Wu et al. reported that the promoter activity of Ezh2 was suppressed by either Oct4 or Sox2 in NIH/3T3 cells in a dose-dependent manner. They also showed that Oct4 and Sox2 were negative regulators of Ezh2, primarily at the post-translational level [18]. On the other hand, Ezh2 could modulate the expression of Sox2 during reprogramming by influencing H3K27me3 on its enhancer region [48]. Moreover, Ezh2 has been shown to contribute to the equilibrium of Nanog-high and Nanog-low states in ES/iPS cells through modifying H3K27me3 at the promoter of NANOG [17]. Taken together, these reports suggest a close correlation between these pluripotency regulators and Ezh2 in mouse embryonic development and cellular reprograming. This may explain the impaired development of embryos to blastocysts in the EZH2-siRNA group. Accordingly, the crucial mechanism that enables EZH2 to support embryonic development may also be involved in adjusting pluripotency-associated transcription factors. Our results showed that knockdown of EZH2 enhanced the expression of apoptosis-related genes, such as BCL-XL, BAX, CASPASE3 and BAD, and accelerated cell apoptosis in blastocysts, which is harmful to embryonic development. This has nothing to do with the microinjection process since there was no significant difference between the nonspecific-siRNA microinjection group and the non-injection control group in levels of apoptosis. The ratio of BCL-XL to BAX reflects cell's capacity to resist apoptosis: the former inhibits apoptosis by blocking cytochrome c, while the latter promotes apoptosis by stimulating the release of cytochrome c for activation of caspases [28,49]. In our study, although BCL-XL and BAX were both down-regulated in EZH2 knockdown parthenotes, the ratio of BCL-XL to BAX also decreased, suggesting a compromised anti-apoptotic potential of cells in these embryos. Similar findings have been reported in spermatogonial stem cells [24], prostate cancer stem cells [27], as well as tumors [50,51], all of which show Alamethicin australia an induction of apoptosis by EZH2 deficiency.