G9a co-localized with histone H3 lysine 9 monomethylation but not dimethylation in a nuclear membrane-dependent manner during mouse preimplantation embryo development

Abstract

Purpose: Histone H3 lysine 9 (H3K9) methylation plays an important role in the regulation of preimplantation embryo development. G9a has been reported to be a major H3K9mono (m1)/dimethylation(m2) methyltransferase and to contain nuclear localization signals. This study was performed to investigate the correlation between H3K9 methylation level and G9a localization when the nuclear membrane undergoes periodic reconstruction in the cell cycle during preimplantation embryo development.

Methods: The fluorescence intensity was examined via immunofluorescence. The mRNA expression of G9awas determined using real-time reverse transcriptase (RT)-PCR. Eight-cell embryos were cultured in KSOM supplemented with nocodazole (0.5 μM) for 12 h.

Results: In this study, it was observed that the fluorescence intensity of H3K9m2 and G9a began to increase significantly from the 4-cell stage and reached the peak at the morula stage (p < 0.001), but the fluorescence intensity declined to 4-cell-stage levels when it reached the blastula stage. We observed a similar pattern when we examined G9a mRNA expression. Once the nuclear membrane disintegrated, G9a and H3K9m1 were not detectable by immunofluorescence; when it was reconstructed, G9a and H3K9m1 had relocated to the cell nucleus. However, no significant change was observed in the H3K9m2 localization or in the G9a mRNA level (p > 0.05) during the whole process. JHDM2A was consistently localized in the cytoplasm irrespective of the presence or absence of a nuclear membrane.

Conclusion: These results indicate dynamic changes in the expression level of H3K9m2 and G9a as preimplantation embryogenesis progresses. G9a co-localized with H3K9 m1 in a nuclear membrane-dependent manner during mouse preimplantation embryo development.

Fig. 1

Dynamic pattern of H3K9m2and G9a expression at different stages during mouse preimplantation embryo development. a. Immunofluorescence image of H3K9m2 and G9a at different stages during mouse preimplantation embryo development. b. Quantitative analysis of fluorescence intensity of H3K9m2 and G9a at different embryonic stages during mouse preimplantation embryo development. The white circle indicates the non-degraded polar body. c. G9a mRNA relative abundance was examined at different stages during mouse preimplantation embryo development by real-time-PCR. The fluorescence intensity of H3K9m2 and G9a at PN stage was defined as “1”, and the fluorescence intensity at other stages were compared with it. All values are presented as the mean ± SD of three independent experiments. Bars denote SD. *P < 0.05;**P < 0.01. Note: P: PN; 2c: 2-cell stage; 4c:4-cell stage; 8c:8-cell stage; M: morula stage; B: blastocyst

Fig. 2

Co-localization of H3K9m2 and G9a in metaphase blastomere cell of a mouse embryo at different developmental stages. The white arrow indicates the metaphase chromosome

Fig. 3

Localization of G9a and H3K9m1/m2 and their correlation with the nuclear membrane. a. Immunofluorescence image of relationship between localization of G9a and H3K9m2 and the nuclear membrane. b. Immunofluorescence image of the relationship between localization of G9a and H3K9m1 and the nuclear membrane. c. G9a mRNA was examined at 8-cell embryos treated with nocodazole by real-time-PCR. Control indicates the untreated 8-cell embryos; nocodazole indicates the 8-cell embryo treated with nocodazole (0.5 μM) for 12 h; nocodazole-2 indicates 8-cell embryo treated with nocodazole (0.5 μM) for 12 h and then cultured in nocodazole-free KSOM medium for another 30 min

Fig. 4

Co-localization analysis between G9 and JHDM2A after the nuclear membrane disintegrated. Control shows the untreated 8-cell embryo; nocodazole represents 8-cell embryo treated with nocodazole (0.5 μM) for 12 h; nocodazole-2 indicates 8-cell embryo treated with nocodazole (0.5 μM) for 12 h and then cultured in nocodazole-free KSOM medium for another 30 min

Fig. 5

Hypothesis for the mechanism of regulation of H3K9 methylation by G9a and JHDM2A during mouse preimplantation embryo development